THE PUREHCATION 0F PEPTIDYL-tRNA FROM RABBIT RETICULOCYTE RIBOSOMES Thesis for the Degree of Ph. D. MECHIGAN STATE UNIVERSITY RONALD CRAIG SLABAUGH 1970 LIBRARY Michigan Static University- THFI‘S'S This is to certify that the thesis entitled THE PURIFICATION OF PEPTIDYL-tRNA FROM RABBIT RETICULOCYTE RIBOSOMES presented bg Ronald Craig SIabaugh has been accepted towards fulfillment of the requirements for Ph. D. degree in Biochemistry /'I / 4 j 9 6 7/41 1/ / ”’2’ 12¢ a) Major professor 0-169 BINDING BY "OAS & SBNS' nnnv nmnrnv INE I HILM‘L'EMWPE ’ f .I 9 3“"; “'3 5 '2‘; ’ £33" . I? I.‘ "33“,) 57".16‘ I. ‘ To Ami? 3f! 313$?“ é: i'fifiu‘ 5'5“ [“Mh‘ 3 j J If flan/{’3 {km 3’3""??? «33‘1"? ‘?>:£m-j an, . A 3 .. 'p 1 ‘5" 7 “'1'". étik stage: 03' “(jg $11.. (groggy; .r’ I ' ti ‘1 ‘ ,4 l f . a Imam at 19:90:. 10m» I}! msm Ir .._..... 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Iii-Q / 2...}- V ,.. . :4. .,. . . «I. . . . . . _ [a a ”vi, 3.471514. ,.r t .x . (a: k“! l: [IQ IL V . )Ii‘r‘m st \ . )Vrvi A proce: ribosomes has moved as we] This constitut from ribosowes | 69] f1 1 t1 ten'Ze peptidy' Iibeled bacter from rabbit re acids in Whme cOnstituted tn RibOSO-"ie t0 solubmze chains WEY‘e Fe: 5130 USed to p a \ J‘ The prom D, ABSTRACT THE PURIFICATION OF PEPTIDYL-tRNA FROM RABBIT RETICULOCYTE RIBOSOMES By Ronald Craig Slabaugh A procedure for purifying peptidyl-tRNA from rabbit reticulocyte ribosomes has been developed. Ribosomal RNA and ribosomal proteins are removed as well as completed and released (from tRNA) globin peptides. This constitutes the first report of the purification of peptidyl-tRNA from ribosomes programmed by endogenous mRNA. Gel filtration on 6-200 Sephadex was used to identify and charac- terize peptidyl-tRNA. The material was labeled with amino acids or 32P- labeled bacterial tRNA in a cell-free protein-synthesizing system derived from rabbit reticulocytes. Peptidyl-tRNA was also labeled with amino acids in whole reticulocytes. Ribosomes isolated from these incubations constituted the starting material in the purification procedure. Ribosomes were treated with LiCl and urea to precipitate rRNA and to solubilize peptidyl-tRNA. Ribosomal proteins and released globin chains were removed by DEAE-cellulose chromatography. The methods were also used to purify peptidyl-tRNA from two additional ribosomal sources, one bacterial (Escherichia coli), and another mammalian source (rat liv- er). The procedure appears to be of general applicability and should provide a method for isolating peptidyl-tRNA from diverse sources. Analysis of 1‘‘C tyrosine-labeled tryptic peptides obtained from purified rabbit reticulocyte peptidyl-tRNA demonstrates that globin chains with completed amino acid sequences are bound to the ribosomes as peptidyl-tRNA. THE PURIFICATION OF PEPTIDYL-tRNA FROM RABBIT RETICULOCYTE RIBOSOMES By Ronald Craig Slabaugh A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1970 Ronald i 1943. He like and seek his ‘ first eightee' "files west of .‘i‘IOH High 50 On the e 30 "work with it ‘dilla‘iette 39335. declare qualifying for 1955, He was E Uni W“ t)’ On SrdOtEd a Nat‘ IEdrS. VITA Ronald Craig Slabaugh was born in Portland, Oregon on February 4, 1943. He likes to tell people that he left Chicago in 1942 to go west and seek his fortune, and was born immediately on arrival. He lived the first eighteen years of his life in Forest Grove, a small town about 25 miles west of Portland. His high school years were spent at Forest Grove Union High School, where he graduated second in his class in 1961. On the advice of his father, he went to college so as to be able to "work with his head, not his hands." He entered the Honors Program at Willamette (pronounced Nill-am’-it, dammit!) University and after two years, declared an English Literature major. He spent his senior year qualifying for a B.A. in Biology, and was graduated.thna cum Laude in 1965. He was admitted to the Biochemistry Department of Michian State University on the basis of his grade point average, not his course work in English Literature. He held a NASA fellowship for three years and was granted a National Institutes of Health Fellowship for his final two years. Ron has accepted a postdoctoral fellowship under the Regional Medical Program of the Department of Health, Education, and Welfare to work in the Office of Medical Education at Michigan State's new medical school. He will probably end up teaching at a medical school, hopefully in the Pacific Northwest. 11' i In hlS ' sweetheart, t ‘ with three be susaort provi: In his junior year at Willamette, Ron married his high school sweetheart, the former Mary Beth Mason, who has subsequently blessed him with three beautiful children, Kristen, Liesl, and Karin. The spiritual support provided by his family is hereby acknowledged. iii :31 or TABLE; 58* 0F FIGURE w'm‘ m.tDUCTION . LUERATURE REV - W Pr m :KIALS AND Compounds Biologica Prep Prep Prel Prel Prep Prep Pres Prep Analytica 3-20 Urea Urea DEAE SUCr Pro: Coon Tryg Pep: High 353173 CharaCt8r G~20 Base TABLE OF CONTENTS Page LIST OF TABLES ......................... VI LIST OF FIGURES ........................ vii LIST OF ABBREVIATIONS ..................... 1x INTRODUCTION . . . . . . . . . . . . . . ............ 1 LITERATURE REVIEW . . . ....... . ......... . . . 4 MATERIALS AND METHODS ..................... 12 Compounds . . ..... . ................. 12 Biological Materials .................. 13 Preparation of Rabbit Reticulocyte Ribosomes ..... 13 Preparation of the Cell- Free System Enzyme Fraction . 15 Prelabeling of Ribosomes in the Cell-Free System . . . 16 Prelabeling Ribosomes in Whole Reticulocytes ..... 16 Preparation of Labeled Globin . . . . . . ..... 17 Preparation of 32P- Labeled E. coli tRNA ....... 18 Preparation of Labeled Rat Liver Ribosomes ...... 19 Preparation of Labeled E. coli Ribosomes ....... 19 Analytical Procedures .......... . ........ 20 G-ZOO Sephadex Gel Filtration . ........... 20 Urea . . . . ....... . ............. 21 Urea Buffers . . . .................. 21 DEAE-Cellulose .................... 21 Sucrose Gradient Analysis . ............. 22 Protein Determination ................ 23 Counting Procedures . ................ 23 Trypsin Digestion .................. 25 Peptide Mapping . . ................. 25 High Voltage Electrophoresis ............. 26 RESULTS . . . . . . . . . . . . ................ 27 Characterization of Peptidyl-tRNA ............. 27 G-ZOO Sephadex . . . . . . . ............. 27 Base Hydrolysis . .................. 3O Protease and Nuclease Digestion ........... 39 Puromycin Effect . . ........... 42 Radioactivity Peak in G- ZOO Sephadex Void Volume . . . 42 Heme- Labeled Ribosomes on G- 200 Sephadex ....... 48 iv Purifica‘ 111 81: DE1 Purifica' Sou Pol Rat i Unsucces. Phe‘ G-Z StUdles .: Try: Page Purification of Peptidyl -tRNA ............... 52 LiCl/Urea Treatment . ............. 52 Bio-Gel P- 10 Filtration Step ............. 52 DEAE- Cellulose Step ................. 6O Purification of Peptidyl-tRNA from Other Ribosomal Sources ....................... 67 Poly U-Directed E. coli Ribosomes .......... 67 Rat Liver Ribosomes ................. 76 Unsuccessful Purification Methods ............. 76 Phenol Extraction .................. 81 G-ZOO Sephadex Chromatography ............ 83 Studies of Purified Peptidyl-tRNA ............. 87 Tryptic Analysis of Rabbit Reticulocyte Peptidyl- tRNA . . . . ................ 87 Chain Separation on CM- Cellulose ........... 91 Fractionation of Peptides by Size .......... 97 DISCUSSION ..... . ..................... 102 LIST OF REFERENCES ....................... 108 F .1. Recove valine Table II. III. IV. VI. VII. LIST OF TABLES Recovery from G-200 Sephadex of the radioactivity of 1“C valine-labeled ribosomes . . . . . ............ Solubilization of peptidyl-tRNA by LiCl/urea ....... Millipore filtration of polyphenylalanyl-tRNA ...... Phenol extraction of labeled rabbit reticulocyte ribosomes ....... . . . . . . . . . . . . . . . . . CM-cellulose chromatography of hemoglobin and peptidyl-tRNA .......... . ........... Attempts to wash peptidyl-tRNA off ribosomes ....... Radioactivity in tyrosine positive spots on a peptide map of C tyrosine-labeled peptidyl-tRNA ........... vi Page 51 55 73 82 88 96 Fig1re U1 15, 3-203 Incor: reticu ."‘ ffqp J‘Lug Aikali Figure 10. 11. 12. 13. 14. 15. 16. 17. LIST OF FIGURES G-ZOO Sephadex analysis of 1“C valine-labeled ribosomes . . Incorporation of 32P-labeled E. coli tRNA into rabbit reticulocyte peptidyl-tRNA . . . . . . . . . . . . . . . . G-ZOO Sephadex analysis of doubly labeled ribosomes . . . . Alkaline hydrolysis of 32P-labeled peptidyl-tRNA ..... Alkaline hydrolysis of 1"C valine-labeled peptidyl-tRNA . . Susceptibility of peptidyl-tRNA to nuclease and protease digestion . ....... . . . . . . . . . . . . . . . . . Sephadex G-200 analysis of control ribosomes and ribosomes incubated in the presence of puromycin . . . . . . . . . . G-200 Sephadex analysis of reduced front material ...... Sephadex G-200 analysis of ribonuclease-treated front material ......... . ............... Sephadex G-200 chromatography of ribosomes labeled with tritiated leucine and 1“C 6-amino-levulinic acid ..... Flow diagram of the purification procedure ........ G-ZOO Sephadex analysis of LiCl soluble material ..... Desalting of LiCl supernatant fraction on Bio-Gel P-IO DEAE-cellulose step . ................... G-ZOO Sephadex analysis of the Buffer II eluate from the DEAE-cellulose step of a purification of 1“C valine- labeled peptidyl-tRNA . . . . . . . . . . . . . ...... G-ZOO Sephadex analysis of the Buffer I eluate from DEAE-cellulose . ........ . ............ DEAE-cellulose column chromatography of a peptidyl-tRNA preparation using unlabeled ribosomes plus ‘ C-labeled soluble globin ...................... vii Page 29 32 34 36 38 41 44 47 62 Finge 13. f'\_) L11 Purif‘ ribos: Purif‘ laseIe 8-230 Analys COFEEH‘ Sucrgg LIST OF FIGURES (cont.) Figure Page 18. Purification of polyphenylalanyl-tRNA from E. coli ribosomes programmed with poly U . ............ 71 19. Purification of peptidyl-tRNA from E. coli ribosomes labeled with 1"C amino acids in viva . . . . . ...... 75 20. G-ZOO Sephadex analysis of E. coZi peptidyl-tRNA . . . . . 78 21. 6-200 Sephadex analysis of rat liver peptidyl-tRNA . . . . 8O 22. Analysis of G-ZOO Sephadex column fractions for protein content . . . . . . .................... 85 23. Sucrose gradient ..................... 9O 24. High voltage electrophoresis of a tryptic digest of 1“C tyrosine-labeled rabbit reticulocyte peptidyl-tRNA . . . . 93 25. Peptide map of ll’C tyrosine-labeled peptidyl-tRNA plus carrier globin .......... . ........... 95 26. Fractionation of peptides from 1"C valine-labeled peptidyl- tRNA on CM-cellulose ................... 99 27. Bio-GeT P-20 separation of 1"C valine-labeled nascent peptides . . . . . . . . . . . . . ..... . . . . . . . IOO viii J- ' C '0 BSA C‘I-CBIIUIOse JEAE-CEIIUIQS ATP BSA CM-cellulose DEAE-cellulose E. coli EDTA GSH GTP 2-ME mRNA PEP poly A poly U POPOP PPO rRNA SDS TCA Tris tRNA LIST OF ABBREVIATIONS adenosine triphosphate bovine serum albumin carboxy methyl cellulose diethylaminoethyl cellulose Escherichia coli ethylenediamine-tetracetic acid reduced glutathione guanosine triphosphate 2-mercaptoethanol messenger RNA phosphoenolpyruvic acid polyadenylic acid polyuridylic acid 1,4-bis{2-(4-methyl-5-phenyloxazolyl)}-benzene 2,5-diphenyloxazole ribosomal RNA sodium lauryl sulfate trichloroacetic acid {tris}(hydroxymethyl)aminomethane transfer RNA All temperatures reported in degrees centigrade ix \' ,_._- Accordi'l 515, amino SIC 1955; Schweet 1959.) On UV— ;=o:yribonuc19C waste of a De tiMIy beginni e: 11., 1360; case sequence Mrection (Sa using ATP by ‘ INTRODUCTION According to the generally accepted mechanism for protein synthe- sis, amino acids are polymerized on ribosomes. (For reviews see Moldave, 1965; Schweet and Heintz, 1966; Lipmann, 1969; Cold Spring Harbor Symp.. 1969.) On these subcellular particles the linear sequence of bases in a polyribonucleotide (mRNA) is translated into the specific amino acid se- quence of a particular protein. Amino acid residues are added sequen- tially beginning at the N-terminal position of the polypeptide (Bishop et al., 1960; Dintzis, 1961), each specified by a 3-base codon in the base sequence of mRNA. Translation of the mRNA proceeds in the 5' to 3' direction (Salas et al.. 1965). Individual amino acids are activated using ATP by the formation of a high-energy ester between the carboxyl group of the amino acid and the 3' hydroxyl group of the terminal aden- osine of a tRNA molecule (Zachau et al.. 1958; Preiss et aZ.. 1959; Hecht et al., 1959). Information needed to decode the mRNA template is provided by the 3-base "anticodon" present in the sequence of the differ- ent tRNA's (Nirenberg et al.. 1965; Soll et aZ.. 1965). The ribosome is thought to provide two binding sites for tRNA mole- cules (Warner and Rich, 1964a; Wettstein and Noll, 1965). Aminoacyl-tRNA binds to the A (or donor) site, in preparation for peptide bond forma- tion. The tRNA bound to the P (or acceptor) site carries the growing peptide chain (Warner and Rich, 1964b; Skogerson and Moldave, 1968). Polymerization occurs through nucleOphilic attack by the amino group of the am’noacyl the C-termina the tRh’A. As chain is trar :ies the A 5‘] other round c sipernataht f LIT-ham. 1956 1368), It can to the riboso 1" this sense temediate ir Peotid; this CEOtral has deait wi Piateg’ and :99” few att 30333 PY‘Ogre . the ( Dart nUCIeit The D 2 the aminoacyl-tRNA on the carboxyl group of the carboxylic ester between the C-terminal amino acid of the nascent peptide and the 3' hydroxyl of the tRNA. As a result of peptide bond formation, the growing peptide chain is transferred to the new tRNA molecule. Peptidyl tRNA now occu- pies the A site, and must be translocated back to the P site before an- other round of synthesis may occur. The energy of GTP and catalysis by supernatant factors are necessary for this translocation (Nishizuka and Lipmann, 1966; Haenni and Lucas-Lenard, 1968; Skogerson and Moldave, 1968). It can be seen from this scheme that nascent peptide chains bind to the ribosomal complex via tRNA during all steps in their biosynthesis. In this sense, peptidyl-tRNA can be considered the active metabolic in- termediate in protein biosynthesis. Peptidyl-tRNA has received a great deal of attention because of this central role in protein biosynthesis. but most of the work to date has dealt with model systems using bacterial ribosomes, synthetic tem- plates, and antibiotics such as puromycin (Lipmann, 1969). There have been few attempts to directly examine peptidyl-tRNA obtained from ribo- somes programmed by natural mRNA's. One of the reasons for this is, no doubt, the difficulty of purifying this hybrid molecule—-part protein, part nucleic acid--from the structural proteins and nucleic acids of the ribosome. The present study has had as its goal, the develOpment of tech- niques for purifying peptidyl-tRNA free from both the chemical constitu- ents of the ribosome and the radioactive contaminants resulting from the methods used to label peptidyl-tRNA. Rabbit reticulocyte ribosomes were chosen as starting material because of t?’ nade in these only two mR‘TA expected to b 1 and S giobi make oossibie Diex protein. 3 because of their availability and the fact that over 90% of the protein made in these cells is rabbit hemoglobin (Dintzis, 1961). Therefore, only two mRNA's and the two corresponding sets of nascent peptides are expected to be present in significant amounts: those for the rabbit a and B globin chains. The availability of purified globyl-tRNA should make possible subsequent studies related to the assembly of this com- plex protein. ‘-.\.‘d. '.'“ T‘ 1"(3- row. A; r: e '. f Gilbert tiie chains a Iateied with IiiTed with he into pol; fifth. ‘ioreo sealed that Iine hl’dT‘Oiy d‘J’i-tRKA was K . LITERATURE REVIEW Gilbert (1963) provided the first demonstration that growing pep- tide chains are bound to tRNA during protein biosynthesis. E. coli tRNA labeled with 32P was added to a preincubated bacterial crude extract pro- grammed with poly U. The radioactivity incorporated from 1"C phenylalan- ine into polypeptide was found to be covalently associated with the 32P tRNA. Moreover, the nature of the bond between tRNA and the peptide re- sembled that between amino acids and tRNA in its stability toward alka- line hydrolysis and hydroxylamine treatment, although this bond in pepti- dyl-tRNA was found to be an order of magnitude more stable than the ester bond in aminoacyl-tRNA. A detailed study of the attachment of peptide to tRNA was made by Bretscher (1963, 1965) using a poly A-directed E. coli cell-free system for the incorporation of lysine into polylysine. tRNA was prepared lab- eled with 1‘‘C in the 3' terminal adenosine. When this ll'C-labeled tRNA was added to the cell-free system along with tritiated lysine, a lysyl- tRNA was recovered carrying both isotopes. The association of 1"C and 3H radioactivity was insensitive to RNase digestion, which yielded a se- ries of oligolysines, each terminating in a single adenosine. Alkaline hydrolysis removed the adenosine, while periodate oxidation before hy- drolysis had no effect on the integrity of the adenosine, thus indicating the bond included one of the ribose hydroxyls of the adenosine. Poly- lysyl-tRNA was more stable to alkaline hydrolysis than lysyl-tRNA (Bret- scher, 1963). RetiCuI when directec an incubatior retained bOur Peptid, suiied by B and added to Iaoeled nasc and characte of the diffe unstabie to Die tth 3711' Pfitidyi-t‘qf 3min051011-13 5 Reticulocyte ribosomes will also synthesize polyphenylalanyl-tRNA when directed by poly U (Hamada et al.. 1968). When ribosomes from such an incubation were dissociated into subunits, the polyphenylalanyl-tRNA remained bound to the large subunit. Peptidyl-tRNA generated from an endogenous E. coli mRNA has been studied by Bresler et a1. (1966, 1968). 32P-labeled tRNA was prepared and added to a crude E. coli extract. The peptidyl-tRNA carrying un- labeled nascent bacterial proteins was separated from the free 32F tRNA and characterized as to base stability. It was found that the majority of the different peptidyl-tRNA's present on these ribosomes were equally unstable to alkaline hydrolysis, but were an order of magnitude more sta- ble than aminoacyl-tRNA. These workers also suggest that the binding of peptidyl-tRNA to ribosomes is more tenacious than that of free tRNA or aminoacyl-tRNA. The stability of the binding of globyl-tRNA to reticulocyte ribo- somes was found by Phillips (1966) to be related to the degree of disso- ciation of the ribosomes into subunits. Under conditions which dissoci- ated all 805 monomers into the 605 and 405 subunits, about half of the radioactivity in the growing peptide chains was released from the parti- cles, with the remainder bound to both subunits. Studies in which both parts of the molecule were differentially labeled suggested that the longest chains are attached to the 605 subunit and bind more strongly than shorter nascent chains. This work corroborates the earlier studies of Schlessinger and Gros (1963) who found that peptidyl-tRNA stabilized the binding of the 30$ and 505 subunits of the bacterial ribosome. Tash- iro and Siekevitz (1965) report similar results for hepatic ribosomes from the guinea pig. To datI tion 0f any I ‘Eat DOIYSCME Nation 0f pr 19738!“ of at! series. They aFCJntS Of De 3f the pestid utiIity more Phenol to ourify pol; 3131] A (RYCM' Hui-tRNA car :teTplate a t me] substrat :eo‘i .utldyI-tranS or suoernatant yer? .50 the binc Din- -( ' - ...OT-:jCITl relec ,D’v'CiII I k , 1956; 30‘!“ ated, fomnin Peeti dVl ~transf WP POIylysyI xenosines , wnic -: 19696) ”3399 bound ‘4 ', ’9 4 ‘: ( poi 6 To date, there has been no report in the literature of a purifica- tion of any natural peptidyl-tRNA. Manning and Meister (1966) report that polysome-bound peptidyl-prolyl-tRNA is a substrate for the hydrox- ylation of proline in collagen biosynthesis and say that they made a number of attempts to purify this material by phenol extraction of micro- somes. They reported, however, that in such extractions only small amounts of peptidyl-tRNA entered the aqueous phase, while the majority of the peptidyl-tRNA was precipitated by phenol, thus exhibiting a sol- ubility more similar to proteins than nucleic acids. Phenol extraction has been successfully used by a number of workers to purify polylysyl-tRNA from bacterial cell-free systens programmed with poly A (Rychlik, 1966; Rychlik et aZ., 1967; Gottesman, 1967; Rychlik et aZ.. 1969; Cerna et al.. 1969; and Bresler et aZ., 1967). This poly- lysyl-tRNA can be rebound to E. coli ribosomes in the presence of a poly A template at least three base residues long. As such, it provides a model substrate for the translocase enzyme and for the ribosome-bound peptidyl-transferase which catalyzes formation of the peptide bond. GTP or supernatant factors were not required for binding, and lysyl-tRNA pre- vented the binding, as did a number of antibiotics (Cerna et al.. 1969). Puromycin released this rebound peptidyl-tRNA as polylysyl-puromysin (Rychlik, 1966; Gottesman, 1967). Lysine from lysyl-tRNA could be incor- porated, forming a polylysyl-tRNA one amino acid longer (Gottesman, 1967). Peptidyl-transferase also catalyzed transfer of the polylysine from the bound polylysyl-tRNA to synthetic substrates, namely 2'(3')-o-aminoacyl- adenosines, which resemble the acceptor end of aminoacyl-tRNA (Rychlik at al., 1969a). The aminoacylated dinucleotide, CpA-Glycine also re- leased bound polylysine from polylysyl-tRNA (Rychlik et al.. 1969a). 7 Jost et al. (1968) used phenol-purified polylysyl-tRNA to demon- strate that reconstruction of 705 bacterial ribosomes from subunits re- quires mRNA (poly A) and peptidyl-tRNA (oligo-lysyl-tRNA). The two tRNA components were first bound to 305 subunits in the presence of poly A. The complex was stabilized by the addition of 505 subparticles. The polylysine in this complex could not be released by puromycin without the addition of GTP and supernatant factors, suggesting that the polyly- syl-tRNA had entered the A, or aminoacyl, site on the ribosome, and translocation was necessary before a peptide bond could be formed. The participation of endogenous peptidyl-tRNA in peptide bond for- mation and translocation has been examined using rat liver ribosomes. Puromycin will release nascent peptides from endogenous peptidyl-tRNA without supernatant factors or GTP (Skogerson and Moldave, 1968). Amino- acyl-tRNA can be bound to these ribosomes if the supernatant transferase I and GTP are present. It is then capable of forming a single peptide bond with the endogenous peptidyl-tRNA. The peptide is now bound through the tRNA in the A site and is not sensitive to puromycin release. Trans- location to the P or peptidyl site can be effected by incubation with transferase II and GTP (Skogerson and Moldave, 1968b; Siler and Moldave, 1969). Haenni and Lucas-Lenard (1968) have shown that the translocation of N-acetyl-phenylalanyl-tRNA on bacterial ribosomes to the puromycin- sensitive site depends on Factor G and GTP. The chemically synthesized polynucleotide AUG(U)6 was used by Erbe and Leder (1968) in an elegant demonstration of the necessity for translocation of peptidyl-tRNA between each cycle of peptide bond formation. This synthetic mRNA directs the synthesis of fMet-Phe-Phe. The addition of GTP and G Factor is necessary for the formation of the tripeptide; only fMet-Phe is made in their absence. Kramer and r3314 without rei a non-enzynatic yla‘enine resid. its nascent cha‘ Peotidyl-‘ in the synthesi 5TWO acid sequ §ested that the 5.:coiolishes u :TWSfer the n, Case the” cor Tan+ ‘ k " 5 On both 5.9. ‘m‘JInetmc 3i N-f0 Fri/Ive absence. Kramer and Klenk (1969) dialyzed rat liver ribosomes to remove mRNA without releasing endogenous peptidyl-tRNA. When poly U was added a non-enzymatic binding of phenylalanyl-tRNA could occur; and this phen- ylalanine residue was capable of forming a peptide bond with the endogen- ous nascent chains. Peptidyl-tRNA is most probably the substrate in the final reaction in the synthesis of a polypeptide, that is, the release of the completed amino acid sequence from the ribosomal complex. Vogel et al. (1969) sug- gested that the ribosomal-bound peptidyl-transferase is the enzyme which accomplishes this release by acting with an altered specificity so as to transfer the nascent peptide to water instead of to aminoacyl-tRNA. They base their conclusion on the parallel effects of a number of mild treat- ments on both release activity (codon and R factor-mediated release of N-formylmethionine from F-Met-tRNA) and peptide bond formation (transfer of N-formylmethionine from F-Met-tRNA to puromycin). Two additional ar- guments support this conclusion. 1) Peptide chain termination measured by release of F-Met is inhibited by chloramphenicol and sparsomycin (Caskey et al.. 1969), antibiotics known to inhibit peptidyl-transferase (Monro and Marcker, 1967; Monro et aZ.. 1969). 2) R factor- and UAG- dependent release of N-formylmethionine occurs only when F-Met-tRNA is lo- cated in the P-site on the ribosome. Similarly, peptidyl-transferase transfers nascent peptides to yield free tRNA only when peptidyl-tRNA is located in the P site. Kuriki and Kaji (1967a,b) have reported that tRNA isolated from polyphenylalanyl-tRNA had very little acceptor capacity for phenylalanine. This suggests that the tRNA in this peptidyl-tRNA may not be phenylalanine < l .e'V .7)— eta. The pol 3. 2:15 ribos: and-tRNA Dena :ertrifugat i or ::n-:iude tha+ 1...... 11% isolation tRNA. The polyphenylalanyl-tRNA was prepared by $05 dissociation of E. coZi ribosomes programmed with poly U. In subsequent studies (Kuriki and Kaji, 1969), it was found that the tRNA obtained from polyphenylal- anyl-tRNA behaved almost identically with tRNAphe upon sucrose density centrifugation, reversed-phase column chromatography, Sephadex gel fil- tration, and methylated albumim kieselguhr column chromatography. When the tRNA was released by puromycin from polyphenylalanyl-tRNA on ribo- somes, it retained most of its acceptor capacity for phenylalanine. They conclude that the tRNA in polyphenylalanyl-tRNA is indeed tRNAphe, and the isolation procedures used somehow result in the inactivation of accep- tor ability. Another approach to the study of peptidyl-tRNA has been the chemi- cal synthesis of specific, short peptidyl-tRNA's. The procedure involves acylation of a purified aminoacyl-tRNA with additional amino acids using appropriate blocking reagents (Lapidot et al.. 1969a). A number of model compounds have been prepared in this manner including gly-phe-tRNA (Lapi- dot et aZ., 1967a); di-val-tRNA, di-phe-tRNA (Lapidot et al., 1968); oli- goglycyl-tRNA, and oligoalanyl-tRNA up to 12 residues long (Lapidot et al., 1969b), and a number of peptidyl-tRNA's containing amino acids not found in proteins (Yankofsky et aZ.. 1970). The peptidyl-tRNA from reticulocyte ribosomes is of particular in- terest because of its role in the assembly of the hemoglobin molecule. The details of this process have been the subject of intense investiga- tions which have produced a large body of information. The peptide chains of globin and protoporphyrin ring of heme are synthesized at approximately equal rates by reticulocytes (Kruth and Bor- sook, 1956). However, association between apoprotein and prosthetic group does "at leased from the 19.53)- FO" 50" 511373;“ Chains I saints at W“ and HinSlOW: 1‘ fine activity have shown tha reticulocyte D iation rate al study (hint e: 31 L chains 1: iated faster 1 Iarger polySO' 10 group does not appear to take place until the completed peptide is re- leased from the ribosome (Felicetti et aZ.. 1966; Morris and Liang, 1968). For some time it was thought that translation of the mRNA for globin chains proceeded at a non-uniform rate, with internal control points at which the rate of translation changed (Dintzis, 1961; Ingram and Winslow, 1966). Hunt et a1. (1968), however, have measured the spe- cific activity of tryptic peptides derived from nascent globin chains, and have shown that ribosomes are randomly distributed on the mRNA in rabbit reticulocyte polysomes. From this result, they conclude that the trans- lation rate along the mRNA strand is also uniform. In a more detailed study (hunt at aZ., 1969) the rate of translation of the mRNA for both a and B chains was determined, and it was found that the a chain is trans- lated faster than the 8 chain. In addition, 8 chains are synthesized on larger polysomes than a chains (Hunt at aZ.. 1968b). In this work the nascent peptides were not purified from ribosomes, but rather ribosomal pellets (105,000 x g) were digested first with RNase and then trypsin. The completed globin chains detected by this procedure were considered contamination of the nascent chains by soluble, released (from tRNA) glo- bin, which non-specifically binds to ribosomes. Colombo and Baglioni (1966) have reported the presence on polysomes of completed a chains at a level of one completed chain per five or six growing a chains. SDS dissociation of labeled ribosomes followed by G-100 Sephadex gel-filtration suggested that these complete peptides were not bound to tRNA (Baglioni and Campana, 1967). No radioactivity was detected in the C-terminal tryptic dipeptide of the a chain of globin (Tyr-Arg) in peptidyl-tRNA from the 6-100 fractionation when the ribosomes were labeled with 1"C arginine. This led these authors to propose a regulatory 1...... neonanism " and SUDSEQ'J :5 subunits and are Sub: 11 mechanism whereby a chains are released from polysomes upon completion and subsequently bind to B chains which are still being synthesized. as subunits are released from polysomes when the 8 chain is completed, and are subsequently assembled into hemoglobin. organic phos; vate kinase ( t‘fies crystal SJZBIlEd by S “5'9 Products «is from Mann Etade POPOP a Store, Iliinoii Tend Nuclear. tion was fPO'il F Mose membrane .21., Keene. New 273:1eiical , Fre I62, supplied t istained from Ph. 3': Ch-cellulose MATERIALS AND METHODS Compounds Phenylhydrazine hydrochloride and p-dioxane were Eastman products, Rochester, New York. Heparin was obtained from Fisher Scientific Co.. Chicago, Illinois; and Nembutal from Abbott Laboratories, North Chicago, Illinois. Labeled L-amino acids were purchased from Schwartz Bio-Re- search Inc., Orangeburg, New York; New England Nuclear, Boston, Massa- chusetts; and Tracerlab, Waltham, Massachusetts. Carrier-free 32F in- organic phosphate was also from New England Nuclear. Tris buffers, pyru- vate kinase (Type II crystalline from rabbit muscle), ribonuclease A (5 times crystallized from bovine pancreas), PEP, and crystalline BSA were supplied by Sigma Chemical Company, St. Louis, Missouri. ATP and GTP were products of P-L Laboratories, Milwaukee, Wisconsin, and reduced GSH was from Mann Research Laboratories, New York, New York. Scintillation grade POPOP and FPO were obtained from either Packard Instruments, Downers Grove, Illinois; Beckman Instruments, Palo Alto, California; or New Eng- land Nuclear. Cabosil powder used in the thixotropic-gel counting solu- tion was from Packard and Liquifluor from New England Nuclear. Nitrocel- lulose membrane filters were obtained from Carol Schleicher and Schull C0,, Keene, New Hampshire. Trypsin (TRTPCK treated) was from Worthington Biochemical, Freehold, New Jersey. Matheson, Coleman, and Bell, Norwood, Ohio, supplied the 1-nitroso-2-naphthol. Sephadex gels and columns were obtained from Pharmacia Fine Chemicals, Piscataway, New Jersey; Bio-Gels and CM-cellulose from Bio-Rad Corp., Los Angeles, California; and Whatman 12 .4 .-.-1'~‘-r its. In JEIE microgranu iiaflow nembran iassachusetts . :7:‘1P1: v 3 1‘. 3 9 1 \ cutaneous injE ‘njettions on tonic saline Nil") (Allen \aC'r.’ and glut 133 :g of he 7 .- .J 1 4‘ :Euiat91\’ l_ a Sha} Tiff-I 13 DEAE microgranular cellulose from Reeve Angel, Clifton, New Jersey. Diaflow membranes and ultrafiltration cells were from Amicon, Lexington, Massachusetts. Biological Materials Preparation of Rabbit Reticulocyte Ribosomes Male New Zealand rabbits were made reticulocytic by four daily sub- cutaneous injections of 2.5% phenylhydrazine. The animals received no injections on days 5 and 6. The phenylhydrazine was dissolved in an iso- tonic saline solution containing 0.13 M NaCl, 5.2 mM KCl and 7.5 mM MgCl2 (NKM) (Allen and Schweet, 1962). The pH was adjusted to about 7 with NaOH and glutathione was added to 10"3 M. On day 7 the animals were given 100 mg of Nembutal and 2,000 I.U. of heparin via the marginal ear vein. Blood was obtained by heart puncture, and the collected blood cooled im- mediately. All subsequent steps were carried out at 4°. The cells were separated from the plasma by centrifugation for 20 min at 2,000 x g in a Sorvall refrigerated centrifuge. The plasma was decanted and its volume recorded. The hematocrit was usually 15-20% with over 80% of the red cells as reticulocytes (Morris, 1964). The cells were washed with a volume of either NKM or the "special saline" described by Colombo and Baglioni (1966), equal to the plasma volume. The special saline contained 0.14 M NaCl, 5.0 mM KCl, and 1.5 mM M9012, and caused less cell lysis during washing than NKM. The cells were first resuspended in a small volume of NKM or saline and filtered through glass wool to re- move any clots or fatty material. The remainder of the NKM or saline was then added, the suspension stirred, and the cells recovered by centrifu- gation. The wash was repeated with a second plasma volume of NKM or 14 saline, followed by centrifugation. A water aspirator was used to re- move the saline and the layer of white cells. The packed cell volume was either measured in a graduated cylinder or calculated from an hema- tocrit determined in a capillary tube. The cells were lysed by rapidly adding four volumes of 2.5 mM MgCl2 and stirring gently for 10 min. Iso- tonicity was restored by adding one volume of 1.5 M sucrose containing 0.15 M KCl. The solution was stirred and the cellular debris removed by centrifugation at 15,000 x g for 10 min. The supernatant solution was decanted and centrifuged for 90 min at 78,000 x g in the 30 rotor of a Beckman L-2 ultracentrifuge. The high speed supernatant was decanted from the ribosomal pellets and frozen until used in the preparation of the enzyme fraction for the cell-free system. The ribosomal pellets were rinsed with 0.25 M sucrose and resuspended in a small volume of 0.25 M sucrose with the aid of a glass homogenizer and loose fitting teflon pestle. Large aggregates were removed by sedimentation at 10,000 x g for 10 min. The suspension, termed "1X ribosomes" (meaning once sedimented), were heavily contaminated with hemoglobin which could be removed by di- lution of the 1X ribosomal su5pension with Medium B (0.25 M sucrose, 0.0175 M KHC03, and 2 mM MgCl2) (Allen and Schweet, 1962), followed by resedimentation at 78,000 x g. This clear pellet was resuspended in 0.25 M sucrose as before, and was termed "2X ribosomes". All ribosome prepara- tions to be used in the reconstituted cell-free system were stored in liquid nitrogen. The concentration of ribosomal preparations as ribo- nuclear protein was determined spectophotometrically at 260 mu using an extinction coefficient of 11.3 for 1 mg/ml (Ts'o and Vinograd, 1961). Concentration of 1X ribosomes determined in this manner was multiplied by 2/3 to correct for hemoglobin absorbance at 260 mu. _._ ___l “I" ' :M~'r.m‘°" . 2:. ~ -‘" .J’Z - Soluble :reoared by i soeed superne than-ed. All in ’ris buffe aTzniu". sul‘ :recioitatin-g This precipj. 33193 by the 337.8111 Dre: t“We 0.32 13-3 “I ”icCl ' 2 we SOILJtic -11.,n‘IIan Su - the 5011 15 Preparation of the Cell-Free system Ehzyme Fraction Soluble enzymes for use in cell-free labeling of ribosomes were prepared by the methods of Allen and Schweet (1962). The frozen high- speed supernatant solution, usually only one day old, was carefully thawed. All steps were done at 4°. The solution was brought to 0.1 M in Tris buffer by the addition of 2 M Tris-HCl, pH 7.5.* Finely ground ammonium sulfate was added slowly with stirring and the protein fraction precipitating between 40 and 70% of saturation (at 4°) was collected. This precipitate was dissolved in 0.1 M Tris HCl, pH 7.5 and reprecipi- tated by the addition of ammonium sulfate to 70% of saturation. The final protein precipitate was dissolved in a minimum volume of a solution con- taining 0.02 M Tris HCl, pH 7.5, 10"3 M glutathione, 10'3 M EDTA, and 10'3 M MgClZ, and dialyzed overnight at 4° against 100 volumes of this same solution. A precipitate which formed upon dialysis was removed by sedimentation at 10.000 x g for 10 min. This enzyme preparation was stored in liquid nitrogen and appeared stable indefinitely at this tem- perature. When it was necessary to add tRNA-free supernatant enzymes to the cell-free system, a protamine sulfate precipitation step was done before ammonium sulfate fractionation. Protamine sulfate was added to 0.4 mg/ml, and the solution stirred for 30 min. The precipitate which formed was removed by centrifugation at 16,000 x g and discarded. *This stock solution of Tris buffer was prepared at 22°. When di- luted and cooled to 4° the pH is 8.1. 16 Prelabeling of'Ribosomes in the Cell-Free System The cell-free system used to prelabel ribosomes was a modifica- tion (Morris, 1964) of that described by Allen and Schweet (1962). The incubation medium contained ATP (1 mM), PEP (2.5 mM), pyruvate kinase (10 ug/ml), a mixture of 19 amino acids missing the amino acid to be added radioactive (0.05 mM each), GSH (0.02 M), KCl (0.05 M), MgCl2 (4 mM), Tris-HCl buffer, pH 7.5 (0.05 M), 1X ribosomes (5 mg/ml), pur- ified supernatant enzymes (at a level determined to be saturating for each enzyme preparation), and a labeled amino acid (0.05 mM). Incubation was carried out at 37° for 5 or 10 min and terminated by adding 20 vol- umes of cold Medium B. The ribosomes were reisolated by centrifugation at 78,000 x g for 90 min. This 2X pellet was resuSpended in 0.25 M su- crose as previously described, and subjected to a low speed (10,000 x g) centrifugation. Occasionally, ribosomes were further washed with an ad- ditional dilution (Medium B) and centrifugation, yielding a 3X prepara- tion. Prelabeling Ribosomes in Whole Reticulocytes Washed, intact reticulocytes were incubated as described by Morris (1964). In later experiments glucose was added and special saline used in place of NKM. The incubation medium contained 0.33 ml of packed, washed reticulocytes per ml, FeSO4 (0.1 mM), Tris-HCl buffer, pH 7.5 (0.01 M), rabbit plasma (0.05 ml/ml), NKM or special saline, glucose (0.2 mg/ml), an amino acid mixture (Borsook et aZ., 1952) missing the particular amino acid used to label, and the radioactive amino acid at the concentration specified in the Borsook mixture. Incubation was at 37° for 10 min, and was terminated by adding 6 volumes of cold saline. The ceIIS “6 Taifles °f E :edures wet" _:id as in 163$ 1 m”. fro-"n the I; soiution w tetogl obi r. and Huehns sodium pnc column fo' 17 The cells were isolated by centrifugation, resuspended in a further 6 volumes of saline, and washed again. Lysis and ribosome isolation pro- cedures were identical to those used to prepare unlabeled ribosomes. Preparation of Labeled Globin Intact, washed reticulocytes were incubated with a labeled amino acid as in the preparation of labeled ribosomes, except incubation time was 1 hr. The cells were washed, disrupted, and the ribosomes removed from the lysate by high-speed centrifugation. The high-speed supernatant solution was dialyzed against 0.01 M sodium phosphate, pH 6.8, and the hemoglobin purified on a CM-Sephadex column by the method of Winterhalter and Huehns (1964). The column (1.8 x 20 cm) was equilibrated with 0.01 M sodium phosphate, pH 6.8. The dialyzed hemoglobin was applied to the column followed by a 100 ml wash with starting buffer. Elution was by an exponential gradient formed by placing 250 ml sodium phosphate, pH 6.8, in a constant volume flask and adding 0.02 M NazHPO4 to it while stirring. Fractions containing hemoglobin were combined and dialyzed against cold deionized water overnight. Globin was prepared from purified hemoglobin by the method of Rossi- Fanelli et al. (1958). The dialyzed globin was added slowly to 20 volumes of cold (-20°) acetone containing 6 mM HCl. The precipitated globin was allowed to settle, and the bulk of the acetone aspirated. The globin was washed twice with fresh acetone at -20° and collected by centrifugation. The precipitate was dissolved in 3 volumes cold deionized water and lyo- philized. The globin was stored over CaSO4 dessicant at -20°. 33w Phosphate F 719'“ ”Iv FeCl: :erol (4 ”II/1) acids (1 9/1)' We added to Browth was "10' 29115 harvest The ceils “‘3' (0.005“). P adding 303 t broken cells C‘JS phase C of ootassiu ate ethano' L. differ 18 Preparation of “P-Labeled E. coli tRNA E. coli strain 8 was grown in 2 liters (four 500 ml flasks) of a low phosphate medium (Gilbert, 1963) containing NaZHPO4 (10‘“ M), CaCl2 (10‘“ M), FeCl3 (10"5 M), MgSO4 (10"3 M), Tris-HCl. pH 7.3. (0.02 M), gly- cerol (4 ml/l), NH4Cl (1 g/l), NaCl (5 g/l), and vitamin-free casamino acids (1 g/l). Two and one half millicuries of carrier-free 32F as H3P04 were added to each flask before inoculation. Incubation was at 35°. Growth was monitored by turbidity (660 mp) in a parallel culture, and the cells harvested in late log phase at an optical density of about 0.8. The cells were collected by centrifugation and washed with Tris-HCl (0.005 M), pH 7.3, containing 0.01 M MgClz. The bacteria were lysed by adding 505 to 1% and incubating for 30 min at 30° (Gilbert, 1963). The broken cells were extracted with buffer-saturated phenol at 4°, the aque- ous phase collected, and the tRNA precipitated from it by the addition of potassium acetate to 2%, followed by two volumes of cold (-18°) absol- ute ethanol. The precipitate was collected and dissolved in Tris-HCl/MgCl2 buffer. The gummy, insoluble DNA was removed by filtering through glass wool. DEAE-cellulose chromatography was employed to isolate the tRNA from the other RNA species present in the preparation (Holly, 1967). The col- umn (1 x 8 cm) was equilibrated with 0.1 M Tris-HCl. pH 7.5 (at 25°), con- taining 0.1 M NaCl. Elution was at room temperature. The absorbance of the effluent was continuously monitored at 254 mp with an ISCO UV monitor. Following application of the sample, the column was washed with starting buffer until well after the absorbance had returned to baseline. The tRNA was eluted with 0.1 M Tris-HCl. pH 7.5, containing 1 M NaCl. Fractions cantaining the 0 ethanol at -18°. and the tRNA whi :recipitate was Pretrztior: of Ribosomes cos of Munro 9' the 1“C anino aere reisoI ate washed by two 1364‘). 19 containing the tRNA were pooled and brought to 2% socium acetate and 67% ethanol at -18°. The preparation was allowed to stand at -18° overnight, and the tRNA which precipitated was collected by centrifugation. The precipitate was dissolved in 0.02 M Tris-HCl, pH 7.5, and stored frozen. Preparation of Labeled Rat-Liver Ribosomes Ribosomes were prepared from the livers of fasted rats by the meth- ods of Munro et al. (1964), and labeled in their cell-free system with the 1'‘C amino acids of a reconstituted protein hydrolysate. The ribosomes were reisolated from the incubation mixture by ultracentrifugation and washed by two additional sedimentations from Medium M (Munro et al., 1964). Preparation of Labeled E. coli Ribosomes The labeling of E. coli ribosomes was done in an "S-30" cell-free system (Forchhammer and Kjeldgaard, 1967) with the assistance of Brian Dunlap of this department. The S-30 extract was prepared from a ur cil- starved culture of E. coli strain 15 TAU (Forchhammer and Kjeldgaard, 1967). Either polyuridylic acid (poly U), or "chloramphenicol RNA" (Slater and Spiegelman, 1966),was supplied as template for the incorpor- ation of 1“C phenylalanine or the ‘“C amino acids of a reconstituted pro- tein hydrolysate. respectively. The incubation proceeded for 5 min at 37°, and was terminated by dilution with cold Tris-HCl (0 005 M, pH 7.5) containing 10 mM magnesium acetate. The ribosomes were pelleted in the ultracentrifuge (165,000 x g for 4 hr) and resuspended in the Tris/Mg++ buffer. They were stored at -18°. BCDTOXT h r drost , l u a: a. an -~d‘° v 5'1; 9.. at 3 :1. .e.» n 1 a: a._ .5 le": F33- 20 Analytical Procedures G-200 Sephadex Gel Filtration Sephadex G-200 (particle size 40-120 u) was swollen in a buffer containing 0.01 M ammonium acetate, pH 5.0, and 0.25% 505. A gel bed of approximately 40 cm was formed in a Pharmacia brand column, K 25/45. The hydrostatic head was never allowed to exceed 15 cm either during the for- mation of the gel bed or during sample elution. The flow rate varied, as the column was occasionally repoured, from 5 - 15 ml/hr. Sample vol- ume was normally 2.0 ml. Sample density was increased by adding 0.1 ml sucrose (1.5 M), and the sample layered onto the nylon sample applicator under the buffer at the top of the gel bed using a syringe driven by a Sage pump. Yeast tRNA* (0.5 mg) was added to each sample as an internal marker in the absorbance profile. This allowed the comparison of data from columns which had somewhat different bed heights or slightly differ- ent fraction volumes. Alternate fractions were collected directly into scintillation vials using a Gilson Model V-10 fraction collector, and counted in 10 ml of a dioxane-based scintillation fluid (Bray, 1960). The absorbance profile was obtained either by measuring the optical den- sity of alternate fractions at 260 mu in a Gilford spectrophotometer, or from the tracing of an ISCO UV monitor (254 mu). Ribosomes analyzed in this manner were first dissociated in 1% SDS for 30 min at room tempera- ture before application to the G-200 column. Elution was carried out at room temperature. *Yeast tRNA kindly provided by Joe Abbate of this department. Ure fzed by 5 tion and ,JVarier a as deion lized at 13:51:“? de :ared fro here aide Sé'ature ‘15 endoth “CI/urea 21 Urea Urea solutions (9 M) were prepared at room temperature and deion- ized by stirring with Amberlite MB-3. The resin was removed by filtra- tion and the urea stored at 4° for periods not exceeding two months (Marier and Rose, 1964). Urea used in the LiCl treatment of ribosomes was deionized as a saturated solution at room temperature and recrystal- lized at 4°. The crystals were collected by filtration and dried by vacuum dessication over phosphorous pentoxide. A stock solution was pre- pared from this Urea to contain 6 M LiCl and 8 M urea. The two compounds were added slowly and simultaneously to keep the solution at room tem- perature to avoid accelerating cyanate formation. (Solubilization of urea is endothermic, while solubilization of LiCl is highly exothermic.) The LiCl/urea stock solution was stored at -20°. Urea Buffers The buffer for the DEAE-cellulose chromatography contained 8 M urea, 0.1 M sodium acetate, pH 5.6, and 0.05 M 2-ME (Buffer I). It was pre- pared from stock solutions of 9 M deionized urea, 5 M 2-ME, and glacial acetic acid. Buffer I was titrated to pH 5.6 at room temperature using 6 N NaOH. All pH measurements were made with a glass electrode immersed directly in the urea buffer. Buffer II was identical except it contained 0.75 M sodium acetate and was titrated with saturated NaOH. DEAE-Cellulose Whatman De-52 microgranular cellulose was used according to the la- boratory manual supplied by the manufacturer.* Seven grams of the *Advanced Ion-Exchange Celluloses Laboratory Manual. H. Reeve An- gel and Co. Ltd., 14 New Bridge Street, London, England. exchanger .i‘e C0 . M 2 Iifinesll r minutes e Lit D’Jiyso~,e i9T9d wif 5rd 10 m' 3'1 to 0 0‘ riiin t1 it’sorbinf CDn‘ “ii-EMS LT r- 0”PIG? 22 exchanger was suspended in 60 ml 0.5 N acetic acid and aspirated to re- move °°2' The slurry was titrated to pH 5.6 using 6 N NaOH, and the "fines" removed by allowing the cellulose to settle for the number of minutes equal to 2.5 times the height of the slurry in cm. The "fines" were then removed by decantation of the supernatant solution. Approxi- mately 60 ml of Buffer II were added and the fining step repeated. This was followed by two subsequent settlings from Buffer I. The total re- maining ion exchanger was poured into a 2 cm diameter column to form a bed of approximately 5 cm. The column was washed with about 100 ml Buf- fer I at a flow rate of 1 ml/min. Sucrose Gradient Ana lysis Linear sucrose gradients from 15 - 30% sucrose were used to analyze polysome content of the ribosome preparations. The gradients were buf- fered with 0.01 M Tris-HCl (pH 7.5 at 25°), and contained 0.15 mM MgCl2 and 10 mM KCl. Samples to be analyzed were adjusted to 1 ml and layered on top of 30 ml gradients. Centrifugation was at 4° for 3.5 hr at 25,000 rpm in the Beckman 25.1 rotor. The gradients were analyzed for material absorbing at 260 mu by puncturing the bottom of the tubes and pumping the contents out through a flow cell (0.5 cm path length) in a Gilford spec- trophotometer. The flow rate of 5 ml/min was maintained with a Buchler peristaltic pump; absorbance was recorded automatically by a Sargent Model SR potentiometric recorder. Gradients used in subunit dissociation studies were 5 - 20% sucrose and contained additions as described in the legends. For radioactivity determinations, the effluent from the flow cell was collected in approxi- mately 1 ml fractions using a Packard Model 231 fraction collector. Radioact f\) ()3 CD :1 14' 3;) I d'iicn . 1.51:) :71. 23 Radioactivity of each fraction was determined by TCA precipitation and counting on membrane filters. Protein Determination Protein concentrations were estimated by measuring the absorbance at 280 and 260 mu and using the nomograph (courtesy of California Corp. for Biochemical Research) constructed by E. Abrams from data of Warberg and Christian (1942). More precise measurements were obtained by the method of Lowry et al. (1951), using crystalline BSA as a reference standard. Counting Procedures Aqueous samples were counted using Bray's solution (Bray, 1960), which contained, per liter: 60 g naphthalene, 4 g PPO, 200 mg POPOP, 100 ml absolute methanol, 20 ml ethylene glycol, and p-dioxane to volume. The 2 ml fractions from the G-200 Sephadex column were counted with 10 ml Bray's solution; other aqueous samples varied in volume from several mi- croliters to 3 ml and were counted with from 5 to 15 ml of Bray's solu- tion. When the G-200 column was run preparatively, the radioactivity pro- file was obtained by spotting 0.1 ml aliquots of alternate fractions on 0.75 in filter paper squares. The papers were dried and counted in a toluene-based scintillation fluid containing 4 g PPO and 50 mg POPOP per liter. Samples in Buffers I and II had to be diluted at least 1:1 with water and counted in an unrefrigerated scintillation counter to prevent precipitation of urea in the Bray's solution. Samples to be counted on membrane filters were precipitated with 5 ml of carrier dried to scintili Ra i‘éttati itntaini .i‘sL, ”lad a T1 2‘- ‘aidne. 24 5 ml of either 5 or 10% (w/v) trichloroacetic acid after adding 1 drop carrier BSA (15 mg/ml). The filters were washed with additional TCA and dried for 30 min in an 80° oven before counting in the toluene-based scintillation fluid. Radioactivity incorporated into protein by the cell-free system was measured in a thixotropic gel scintillation mix after preparing the sam- ple as described by Casjens and Morris (1965). Bovine serum albumin (15 mg) was added to each sample followed by precipitation with 5% tri- chloroacetic acid. The precipitate was washed by resuspension and resed- imentation in 5% trichloroacetic acid. The resultant pellet was dissolved in 0.5 ml of 1 N NaOH, washed once more, and then suspended in acetone containing 0.1 N HCl. The precipitate from the acetone step was washed with a mixture of acid-acetone and ether (2:3), and finally with ether alone. The resulting powder was transferred to a glass counting vial with the aid of 0.5 ml of 1 N NaOH. When the protein powder had complet- ely dissolved, 15 ml of thixotropic counting mixture was added and the contents of the vial were shaken vigorously. The counting mixture con- tained 7 g of PPO, 150 g of POPOP, 50 g naphthalene and 35 g of thixotro- pic powder dissolved in 200 ml of toluene, 30 ml of ethanol, and 800 ml of p-dioxane. Radioactive carbon on Whatman 3 mm paper used for peptide mapping or electrophoresis was measured by immersing the paper strip directly in tol- uene scintillation fluid. When tritium was the radioactive label, the paper strips were placed in counting vials and covered with 1 ml 1% (v/v) formic acid. The vials were heated for 3 hr at 60° to extract radioactive peptides. After cooling, 15 ml Bray's solution was added. added to 0. :l) as a pH 37-0 (Kitche Eric-t or ei in q, . ble C Prise. I " 5 I'Iei‘e Us”; 25 Trypsin Digestion Globin samples to be treated with trypsin were first dialyzed overnight at 4° against deionized water. Ammonium bicarbonate was added to 0.2 M along with several drops of phenol red (0.1% in 20% ethan- ol) as a pH indicator. Trypsin (Worthington TRTPCK treated) was added at a weight ratio of 1:100 or 1:50 and the sample incubated for 2 hr at 37° (Kitchen and Easley, 1969). Digests were lyophilized before finger- print or electrophoretic analysis. Peptide Mapping Samples of tryptic digests containing 1.5 - 2 mg in 20 pl of 0.2 M NH4HC03 or 1% formic acid were applied to Whatman No. 3 mm paper. Pep- tides were separated two-dimensionally by the method of Katz, Dreyer and Anfinsen (1959), as modified by Kitchen et al.(1968). Chromatography buffer (n-butanol:glacial acetic acidzdeionized water, 4:1:5), was pre- pared in a separatory funnel. The lower (aqueous) layer was placed in the bottom of the chromatocab to equilibrate the paper, and the upper (butanol) phase was used to elute the sample. Papers were equilibrated in the closed chromatocab for at least 2 hr before adding the butanol phase. The chromatogram was developed for 16 hr. After drying, the pa- pers were trimmed, turned 90°. and placed on the lucite rack of a Gilson Model 0 High Voltage Electrophorator. The paper was wetted with the pH 4.8 electrophoresis buffer (Kitchen et al., 1968) (1.25% pyridine, 1.25% acetic acid) before electrophoresis for 1.75 hr at 2,000 volts. Peptides were visualized by dipping through buffered ninhydrin (Easley, 1965) con- taining 0.3% (w/v) ninhydrin in acetone which was 1% (v/v) in glacial acetic acid and 1% (v/v) in pyridine. Tyrosine-containing spots were identified 115$ dipped 2d dipaed Tyrosine-ct termed 11 26 identified using a 1-nitroso-2-naphthol stain (Easley, 1965). The paper was dipped in a 0.1% (w/v) solution of this compound in acetone, dried, and dipped through acetone containing 10 ml concentrated HNO3 per 100 ml. Tyrosine-containing spots were cut from the paper and radioactivity de- termined in 15 ml toluene scintillation fluid. High Voltage Electrophoresis Labeled peptides were sometimes separated with only the electro- phoresis part of the peptide mapping technique. The 100 pl sample was applied to the paper as a 2 cm streak, with an adjacent 5 pl guide spot. Authentic standards of Tyr-Arg and Tyr-Hist were run on the same paper. The portion of the paper containing the guide spot and the standards was cut off and stained with buffered ninhydrin followed by the 1-nitroso- 2-naphthol staining procedure. The sample lane was cut into 0.5 cm sec- tions which were counted with 10 ml toluene scintillation fluid (for 1‘’C), or Bray's solution (for tritium) (See Counting Procedures). Li“: I :35 oft ... "J l D RESULTS Characterization of Peptidyl-tRNA 0-200 Sephadex Gel filtration on Sephadex G-200 has been used to identify peptidyl- tRNA. Quantitation was obtained from radioisotope measurements since the mass of nascent protein relative to the mass of the ribosomes is very small, and nascent chains can be labeled under conditions which do not label ribosomal structural proteins. When ribosomes labeled with a ra- dioactive amino acid, either in intact reticulocytes or in the cell-free system, were dissociated with SDS and applied to a Sephadex G-200 col- umn as described under Methods, a characteristic (Gilbert, 1963; Bresler et al., 1966) peak of radioactivity appeared (Figure 1). The identifica- tion of this peak as peptidyl-tRNA has been accomplished in a number of ways. First, it represents incorporation of amino acid label into a macro- molecule. Although the elution profile of radioactivity shown in Figure 1 was obtained by measuring the total radioactivity present in each frac- tion with Bray's solution, an almost identical pattern was obtained when the fractions were precipitated with TCA and counted on membrane filters. The only difference in the radioactivity profile was the disappearance of the last peak labeled "free amino acids“ in Figure 1. The position in the elution profile of the peak labeled "peptidyl- tRNA" in Figure 1 suggests a molecule smaller than rRNA and other excluded species, but larger than the tRNA used as a marker. A number of experi- ments confirmed the fact that this characteristic position on G-200 27 28 .ouoxpoz.mam .mmsomonwg we cowumgmqmga on“ can mgaumuoga :5: -_oo as» to ”Plasma tau .mmEOmonwL anmnmpumcw_m> 0:” mo mpmxpmcm xwumgqmm oomuw .H «camp; 29 0—0 (rim 092) eouquosqv v.0 QC N._ 8952 8:08.... 100. 00 00 ON 00 00 HDTV On :6 {I v“ 4 ; 0 O - O 7.4% TQ.Fm ego m—Pmu not pmucmEPLmaxu . Us” sup: umpmnmp use mo mwmxpoguac mcwpmxp< .m mgzmwu aouquosqv 5'. '0. N. -: 38 0—0 ("lu-l 092) I TI ] nwlsnoliailwunltunlwunlnnulsfl 40‘ Q ~ . ~ ~ 4 ~ 52:52 8:08... Om _ .( . — -! «\1 .Eu Menorah H.253. ”Danae fiflun Ems—>238 m mcgmwu CON (3 CD Us” cup: umpmnop APE m.ov och .megms o ummmoa new mom NH ;u_3 vmumPuommwn wed: Amadeus: mwmv mchm> as“ now: umanmF mmsomonwm . Us_ gap: sweeps: cw =m>pm owes» mam: mco_uwo:oo cowpmasucu .cms m umgpw ms» Low pmuwpcmow mm: mm—QEmm oz“ use we cowuonaucH .cwoaeogaa do mucmmmga on“ cw wagonsucw mwsomonwg ucm mmsomon_g Pogucou ac mama—mew oomuw xmumsamm .N mgzmwa 44 —— (mu 1793) aouoqmsqv 9. '9. “E "I ‘f. '0. 9! " T l l l l l l [‘9 Id <12) 3 _____...< ’ 4o 50 60 7o 80 so . Fraction Number 3C) ---~--<------ 4, <.' if 4”“, IE <1 mug/<1— .— -~<1\ , ‘-~<1-- j C 'o' "2. t: E C E 8 E I l l l 1 l l g l A 1 §, § § § 2. 2 § v-—v wdo 3171 2C) "3 Figure 7 45 treated ribosomes (Figure 7). According to the accepted mechanism for puromycin action, nascent chains should be released from the ribosome and thus would not appear in this experiment. One possibility for aggregation of peptidyl-tRNA molecules is di- merization or polymerization through disulfide bond formation between sulfur-containing bases in the tRNA or cysteines in the protein. Treat- ment with a reducing agent would separate such complexes and they should appear at the peptidyl-tRNA position on G-200 Sephadex analysis. Figure 8 shows that this did not occur, but that the material was again excluded from the gel under these conditions. Ribonuclease treatment should shift the excluded radioactivity from the void volume of the G-200 Sephadex column to a later position in the elution profile upon reanalysis on G-200, if this radioactivity is present as peptidyl-tRNA. The excluded peaks from several G-200 columns were pooled, dialyzed extensively against 0.1 M ammonium acetate, pH 7.0, to remove SDS, and incubated with ribonuclease. Following incubation, the sample was applied to the G-200 column. The results, shown in Figure 9, indicate that the radioactive material was not digested by the RNase, at least not sufficiently to reduce the size below that excluded by G-200 Sephadex. The rRNA present in the sample provides a control: The A25° material (rRNA) originally present in the excluded peak has been digested by the RNase and now appears (as nucleotides) at the final volume of the column in Figure 9. In several identical experiments where the SDS was apparently not sufficiently removed by the dialysis procedure, this A25° material was excluded a second time, indicating no RNase digestion. It should be mentioned that 32F tRNA-labeled ribosomes also gave an excluded peak on G-200 (Figure 2). Presumably this would be RNase 46 .oomnu xmuosamm co mama—ace maommn umuoo Loxgos on» .o.u :a .mpmumoo Ezwcoesm z H.o pmcmmmm mwmx_owu umummamc ma um>oemg mom msu can cmpooa mm: mesapou oomuw pogm>mm sou» xmma umuapuxm one .megmums pcogm nuance» mmmm_o=:on_g mo mamzpocm oomuw xmumzaom .mzum mo mucmmmga mg» cw oomuu co mamxpmcmmg mgomwn NZTN _s\s: ooH gut: u;m_:gm>o cognac» mo: mmsomoapg nmpmnmp -mcw_o> 0:" mo copumcowuuoga csapou oomuw m sou; megmpoe umuzpuxm ac m_asmm FE N < .mzum :5 om new mam am~.o .o.m :a .mgopmum szpcoEEo z Ho.o saw: umaognw_w:am mm: czapou xmuczamm oomuw one ._mwgmume pcoca umuzumg mo mrmxpmcm xmumcamm oomuo .m mgzmwa .m manor; m ogzmwa 89:3,. Scoot o . . . 2. 8 . x .o on N8 0. on w i,,, /.\__ W. «.91 I/ ._ . .. __ w. W QQI / _ .— _ a 42m. " . m a u. _. . m 0.01 C. 1% u m . ,_. a- u o._ m mgamwa v 8232 562". m. 8. cm on 2. 8 o... ow an on o. _ _ _ _ _ filhl _ ._ _ _ q I.- w . Ls m no .T. 1 9 0 ) to... m 7a m «T <2m.\o . n _ n. t 48 sensitive, and could represent rRNA contamination of the 32F tRNA pre- paration. It could also be tRNA bound, perhaps by duplex formation, to the unlabeled rRNA from the rabbit ribosomes, or a small amount of con- taminating E. coli DNA. The 32P tRNA analyzed alone on G-200 also showed radioactivity at the void volume of the column. The significant finding is that RNase does not release amino acid labeled material as would be expected for peptidyl-tRNA. Finally, labeled globin and hemoglobin analyzed on the G-200 column also gave rise to radioactive material in the void volume. This was the case for both amino acid-labeled (Figure 1), and for heme-labeled (Figure 10) material. It was concluded that the excluded material was not peptidyl-tRNA, and probably represented aggregated protein or nucleic acid. Table I in- dicates the proportion of the label in each radioactivity peak for a typ- ical G-200 fractionation of 1‘’C valine-labeled ribosomes. Peak I seldom exceeded 10% of the cpm applied to the column. Heme-Labe led Ribosomes on G-200 Sephadex In collaboration with Morris and Liang (1968) the G-200 Sephadex method for identifying peptidyl-tRNA was used to demonstrate that the heme portion of the hemoglobin molecule does not become associated with the polypeptide chains during translation. Ribosomes labeled in a cell- free system (Liang, 1966; Morris and Liang, 1968) with 3H leucine and 1‘*C o-amino levulinic acid (a heme precursor) were analyzed on G-200 Sephadex 11) determine whether or not the 1"Clabel would appear in the peptidyl- ifl2NA.peak. The results, in Figure 10, show a ll’C peak which corresponds 11) completed and released globin, but ll*C radioactivity is absent from the peptidyl-tRNA peak. 49 Adv £5,532 “.3821? new Adv 23.5.35 33%: load Low umnxpmcm mm: mcowuumgw muwcgmppm wo posowpm we m.o a can Anuv umcwsgmumu mm: as cow an mocongomn< .oomuc co mwmAchm meowwn venue no; wp «wage; as _ ace mam NH sow; nopawooaawa ago: Ass my mascaoawt sopoaa_ age .Amom_v mesa; use mwggoz An umegowgma mm: mcwu=m_ uwpmnmpuzm veg cwum uwcwpa>mpnocwsmuo capo; -m_-os~ saw; mmeomonwg muxoopauwuog “wanna wo :owpmaaucH .uwum uwcw_:>mpnocwsmum as" can mcwuzmw umumwpwgu sow: umpmnmp mmsomoawg wo zsgmgmouoeoggo com-u xmumcaom .oH atamwa 50 ital. '.-.....v )z-o‘ x ulna/810000) W011. ‘ fl 1 ‘ — rfi 1 I l '1. 0‘0 '0 G "’ N m o a...» (mm/swoon) woedvomovu E 2.0" .‘ i 00 I O Q’ m LU (D 2 D Z “,4" Z “’a",’ g - -—-—--' 5 fit '4 z ————————————— ‘ --------- ‘_~~‘ . E 4 4 .34 . 5 -3 ’7 v \‘ 8 .— Gr 9- a Q) ‘5 in o '0. .0; <5 E O O 03 ._ I [6 N 1 l - l l TM 092) BONVBHOSBV I- 51 Table 1. Recovery from G-ZOO Sephadex of the radioactivity of 1‘0 val- ine-labeled ribosomes. Labeled ribosomes were dissociated with 1% SDS and applied to the G-200 Sephadex column (see.Methods). Aliquots (0.1 ml) of each fraction were counted with 2 ml H20 and 15 ml Bray's solution. Cpm for the frac- tions within each peak were summed. I, II, and III correspond respec- tively to the excluded, peptidyl-tRNA, and free amino acid peaks of Fig- ure 1. Peak cpm % of Applied cpm I 11,490 7.4 II l20,520 77.4 III 14,770 9.5 94.3* *Total recovery was quantitative, with the difference between 94.3 and 100% in tubes between the major peaks. 52 Purification of Peptidyl-tRNA The procedure resulting from these studies which successfully pur- ified peptidyl-tRNA free of rRNA, ribosomal proteins, and labeled globin, is diagramed in Figure 11. All steps were carried out at 4°. LiCl/Urea Treatment The stock 6 M LiCl. 8 M urea solution (see Methods) was added to a ribosomal suspension (2 - 20 mg/ml) to give a final concentration of 3 M LiCl and 4 M urea. Sodium acetate buffer, pH 5.6, and 2-ME were both added to 0.05 M. The sample was allowed to stand at 4° for 16 hr. This treatment quantitatively solubilized ribosomal proteins and tRNA while quantitatively precipitating rRNA (Mathias and Williamson, 1964). The precipitate was removed by centrifugation at 10,000 x g for 10 min. The rRNA pellet was washed twice by resuspension in the buffered 3 M LiCl/4 M urea, and the washes combined with the initial supernatant fraction to provide quantitative solubilization of the radioactivity in the ribosomal sample (Table II). Sephadex G-200 analysis of the LiCl/urea soluble mat- erial confirmed removal of the rRNA and demonstrated the integrity of the peptidyl-tRNA material during the LiCl dissociation step (Figure 12). Bio-Gel P-10 Filtration Step The sample was desalted by passing the supernatant fraction from the LiCl step over a 1.9 x 30 cm column of Bio-Gel P-10 equilibrated with Buf- fer I (see Methods). The macromolecules were eluted at the void volume of the column in Buffer I, while contaminating labeled amino acids were re- moved with the LiCl. The elution profile of the P-10 column appears in Figure 13. The LiCl was well separated from the excluded volume even with samples as large as 20 ml. Figure 11. 53 Flow diagram of the purification procedure. Buffer I con- tained 8 M urea, 0.1 M sodium acetate, pH 5.6, and 0.05 M 2-mercaptoethanol. Buffer II was identical, except the sodium acetate concentration was increased to 0.75 M. For preparation of these buffers, see Methods. 54 Purification of Peptidyl-tRNA . Ribosomes (labeled in nascent peptide chains) 3 M LiCl 4 M urea 16 hr 40 F‘T ll Ribosomal proteins rRNA tRNA, SS RNA (?) peptidyl-tRNA Bio-Gel P-10 in Buffer I FTT l Excluded volume Total volume macromolecules any contaminating labeled amino acids DEAE-cellulose in Buffer I T l Buffer I wash Buffer II wash globin, ribosomal peptidyl-tRNK proteins tRNA aa-tRNA Fl'Qure 11 55 Table II. Solubilization of peptidyl-tRNA by LiCl/urea. 1"C valine-labeled ribosomes (see Methods) were treated with 3 M LiCl/4 M urea as described in the text. Aliquots of the supernatant fraction and each of the washes were counted as well as the rRNA pellet which remained. Fraction cpm % of Total Supernatant 22,6l7 9l lst wash of ppt. 1,597 6 2nd wash of ppt. 270 l Total soluble 24,484 -9§—- rRNA 392 l.6 56 .mgzcmuoga caspou com mwoxumz mom .cE:Pou xwvmsamm oomuu on“ :0 nm~xpmcm can .mom wmm.o ucm o.m :3 .mumpmum Ezwcossm z Ho.o newcwmu -cou gmmtsn m2» zuwz Fe e on umpapwn mm: APE Hy mmsomonwg umpmnm_umcwpm> Us" soc» cowuumge penumcgmaam powJ one .megmpms anapom Fum4 mo mmmxpmcm xmvmsamm oomuw .NH mezmwa 57 (rim 1792) aouquosqv "2N“. Ill Ioo' s. (D .0 E :3 I Z 46‘ C .2 +- 0 O i O Q’ 0 l0 0 N 9 N G.) S. 8» 1 1 J L; O O 0 IO N - v—-v undo 3w 58 avoid: 8m mgsumuoga caspou mg» mo mppmumu Lou .Pe u.~ xpmumswxognam mm: waspo> coppumgm .mmeomon_g umpmnm—umcwmogxu 9.H soc» pmwgmuus wpnapom mac: 2 «\Fowg z m «ca mo FE ~.o mm: quEmm use .ogua Pouuowm co cowuumge acmpmcgmaam _uw4 mo mcwu_mmmo .mH me=m_a 59 ow Om mmmEDZ 2078de ON — mH weaned OO. CON 00m .LOTlOI'lV In 9| Jed was) 0... f -' I J- nu! 60 DEAE-Ce ZZu Zose Step The desalted material from the Bio-Gel P-10 column was applied directly to the equilibrated DEAE-cellulose column with a peristaltic pump. Radioactivity eluting with the sample application and subsequent wash with Buffer I represents the globin contamination (Figure 14). The extent of globin contamination varied with different ribosomal prepara- tions from 2 to 20% of the total radioactivity applied to the column. “1 In the column shown in Figure 14, 2% of the cpm applied eluted with Buf- . fer I, and about 75% with Buffer II. The peptidyl-tRNA is bound to the DEAE-cellulose via the phosphate i charges in the tRNA portion of the molecule under these conditions of pH and ionic strength. Buffer II, containing 0.75 M sodium acetate, re- leased the peptidyl-tRNA (Figure 14). Radioactivity eluting in this fraction represented from 65 to 80% of the total radioactivity applied to the column. Figure 15 shows the G-200 Sephadex analysis of the peptidyl- tRNA from the DEAE-cellulose column. When the Buffer I fraction from DEAE-cellulose was analyzed for a preparation of peptidyl-tRNA labeled with 1"C tyrosine, the profile shown in Figure 16 was obtained. A peak near the tRNA marker is no doubt glo- bin, while higher molecular weight species, probably aggregated globin or hemoglobin, are seen between the globin peak and the void volume. In ad- dition, there appears to be a tyrosine peak at the final volume of the column (fraction 90). This would suggest that not all the tyrosine was nemoved by the Bio-Gel P-10 step. No tyrosine was seen, however, in the (3-200 analysis of the Buffer II (peptidyl-tRNA) fraction of this same Preparation (not shown), demonstrating that residual amino acids are re- moved by the DEAE-cellulose step. This was confirmed by passing individual 61 .mwoxuoz.cw vmavgummu mm um~xpmco mew; copuumgw sumo yo muoaum_< .HH Lowesm mcwapanm mgoema H gowmzm FE omH sup: cosmos mm: csa—ou use .Amwoxeoz.mmmv csapoo mmopappousm 0..H we mFaEmm < .ampm mmopzppwouu 9.. to coppau_ewe=a a to amen mmopappmuumwumuwpcm=a no: xgv>Paumo_umg eo cowucmumm .mcwensme mus: 3o—mmwo a co vmumgucmucou mm; euumopumg Low vo~xpmcm mew: meowuumgm caspou m80 0:26.. H l 0 N meccsm Hu 3 o m m _o 3 3.02.0: 2:309. U) N. mm, a... ._ ii a am we .Om o 0 .mL A’I‘ LE 69 Figure 18. 70 Purification of polyphenylalanyl-tRNA from E. coli ribosomes programmed with poly U. A. G-200 Sephadex analysis of the labeled ribosomes. Approxi- mately 0.1 mg of ribosomes (see Methods) were dissociated with 1% SDS and analyzed on G-200 Sephadex as described under Methods. B. DEAE-cellulose step in the purification of polyphenyl- alanyl-tRNA. Buffer I eluted 1% of the radioactivity while Buffer II eluted 38%. C. G-200 Sephadex analysis of purified polyphenylalanyl-tRNA. A 0.5 ml sample of the Buffer II eluate was diluted to 2.0 ml with the SDS buffer and the yeast tRNA marker (0.5 mg) added before application to the G-200 Sephadex column. .:I. . Inl- 71 IEE come 8:38.34 4.. 5 2 J - _ _ _ _ .oo 60 7O 80 90 50 ‘ ‘ ‘ ‘ ‘ ‘ ‘ 5‘5‘ “ ‘ “ “ 4o Fraction Number IO 20 3O l2 4. 0 I2 Fraction Number 20”Fin—Astana I———-9<————Buffer II——-> IGOO- 200 800 - 400- --.Ian Q! lee some meoncomnq 4.. 3 2 J m d‘fiA w AU 8 A N m ml. a mb ............ .m \ ‘l .r cccccccc mm ........... F m 0 2 to C m _ WPJ%FW . npvk m m 4 w w 3 2 I Figure 18 72 DEAE-cellulose as described above. The elution profile of the DEAE-cel- lulose step appears in Figure 188. Only 1% of the radioactivity applied to the column was eluted in Buffer I, while in Buffer II, 38% was eluted. The remaining 60% presumably remained on the column. The Buffer II eluate was analyzed on G-200 and shown to be peptidyl-tRNA (Figure 18C). It was reasoned that perhaps the low yield resulted from a low sol- ubility of polyphenylalanyl-tRNA in these buffers due to the hydrophobi- ”3 city of the polyphenylalanine portion of the molecule. An experiment to ;i test this possibility was performed, and the results appear in Table III. Aliquots of the material pooled from the P-10 column and of the Buffer II F 5’ eluate from DEAE-cellulose were counted before and after filtration through a millipore filter. The results in Table III show that the poly- phenylalanyl-tRNA was retained on the filter. The loss on DEAE-cellulose might therefore be attributed to a filtration by the column bed and not ion exchange. It was concluded that the procedure reported here is not 'the method of choice for preparing polyphenylalanyl-tRNA. An attempt was made to label the nascent peptides of bacterial ri- toosomes programmed by endogenous mRNA. Two approaches were taken: 11) labeling bacteria during growth with 1"C amino acids, and 2) labeling vvith 1"C amino acids in an S-30 bacterial extract programmed with "chlor- 21mphenicol RNA."* The preparation of these ribosomes is described under Methods . The results of the purification of peptidyl-tRNA from the E. coli r‘ibosomes labeled in vivo appear in Figure 19. Figure 19A shows the G-200 chofile of the isolated ribosomes. It appears that molecular species *A preparation of RNA with high template activity obtained from baCteria exposed to the antibiotic, chloramphenicol (Slater and Spiegel- ma n , 1966) . 73 Table III. Millipore filtration of polyphenylalanyl-tRNA. Aliquots of the pooled material from Bio-Gel P-10 and the Buffer II eluate from DEAE-cellulose were filtered through a Millipore filter. Radioactivity was measured before and after filtration as well as that present on the filter. All samples were brought to 1 ml with water and 2 drops carrier BSA (200 ug/ml) were added plus 5 ml 10% TCA. The sam- ples were poured over membrane filters and counted as described under Methods . Sample cpm Total cpm % Recovered P-10 0.1 ml before filtration 115 1620 ---- duplicate l02 0.1 ml after filtration 0 -.-- 0% duplicate 0 ---- 0% filter 3030 3030 187% DEAE-II 0.1 ml before filtration l5 520 ---- duplicate ll 0.5 ml after filtration 0 ---- 0% duplicate 0 ---- 0% filter 372 71% Figu re 19. 74 Purification of peptidyl-tRNA from E. coli ribosomes labeled with 1“C amino acids in vivo. A. G- 200 analysis of the labeled ribosomes. Approximately 2 mg E. coli ribosomes from cells grown in the presence of 1‘‘C amino acids (see Methods) were dissociated in 1% SDS and analyzed on G- 200 Sephadex as described under Methods. B. DEAE-cellulose step of a purification of peptidyl-tRNA from the above ribosomes. Buffer I eluted 65% of the radio- activity applied to the column, with 10% eluting in the Buf- fer II fraction. C. G-200 Sephadex analysis of the Buffer II eluate. The Buffer II fraction from the DEAE column shown in B was con- centrated by ultrafiltration on a Diaflow UM-Z membrane. 1 ml of the concentrated material was diluted 1:1 with the SDS buffer, yeast tRNA marker (0.5 mg) added, and the sample applied to the G-200 column. 75 13.. $9 «23.82 n~.s 2» 9_.J q 4 III II n............................. ICKD 4“) 5C) 6!) 7!) EM) ‘9C> Fraction Number l() 2!) E“) Iooo eoo uffer II—‘f Ll -—Buffer I D If 9 l2 4. C) a/é/ //////r/ mLI - P m .6 .m as ...e8 02 .% K5 2C) Fraction Number 12 as .9 an 0‘ Ia IO zoaoiosoeoroeoeo C) IEE 0&8 859634 Fraction Number Figure 19 76 other than peptidyl-tRNA were labeled by the mixture of HC amino acids during the growth of the cells. RNA could become labeled by amino acids through purine and pyrimidine biosynthesis, hence the label in the void volume (rRNA). Figure 198 shows the distribution of radioactivity on the DEAE-cellulose column of this preparation. Buffer I elutes ribosom- aal proteins, which would also be radioactive under these labeling condi- tions. Figure we is the G-200 profile of the Buffer II fraction from a the DEAE-cellulose column. Buffer II eluted material from G-200 Sepha— “ dex at the peptidyl-tRNA position. The major radioactive peak, however, J i s closer to tRNA, which would also be labeled. J The peptidyl-tRNA fraction from the bacterial ribosomes labeled in tzhe S-30 cell-free system (see Methods) was purified and analyzed on G-200 Sephadex. Figures 20A, 8, and C show the G-200 profiles of the ribosomes, t;he purified peptidyl-tRNA, and base hydrolyzed peptidyl-tRNA, respect- i vely. Overall recovery from DEAE-cellulose was 70%, with 90% of the czounts recovered in the peptidyl-tRNA (Buffer II) fraction. Rat Liver Ribosomes Rat liver ribosomes prepared as described under Methods were sub- J’ected to the purification procedure outlined above. Figures 21A, 8, and C3 show the G-200 Sephadex analyses of these ribosomes, the purified pep- t»‘idy'l--tRNA, and base hydrolyzed peptidyl-tRNA, respectively. Recovery f'V‘Iom DEAE-cellulose was about 70%, with 90% of the recovered counts in ‘tiie Buffer II fraction. Unsuccessful Purification Methods As the lack of literature verifies, peptidyl-tRNA from ribosomes D'"1011rammed by natural mRNA's has, to this point, resisted purification. 77 Figurma 20. G-200 Sephadex analysis of E. coli peptidyl-tRNA. A. Approximately 0.5 mg E. coZi ribosomes labeled with HC amino acids in a cell-free system (see Methods) were disso- ciated in 1% SDS and analyzed on G-200 Sephadex as described under.Methods. B. Purified peptidyl-tRNA. 2.0 ml of the Buffer II eluate from DEAE-cellulose was brought to 1% in SDS, and 0.01 M in ammonium acetate. Yeast tRNA marker (0.5 mg) was added and the sample analyzed on G-200 Sephadex as described under Methods . C. Alkaline hydrolysis of purified peptidyl-tRNA. 2 ml of the Buffer II eluate from DEAE-cellulose was brought to pH 9.0 with 6 N NaOH. Yeast tRNA marker (0.5 mg) was added and the sample incubated 1 hr at 37°. After incubation, the pH was lowered to 5.0 with HCl and the material analyzed on G-200 Sephadex as described under Methods. 78 line vmmv 4.3.2.1. moze eo muoaame He H :H cowpmeucmucoo cwmuoea on» .mwoeemz Love: conHLUmmu me xmueznmm com-o op umHHaam new mmeomon_g umHman-mcHHe> 0..d we on omH op venue mew: Ame oHV mmeom -onHe omHmancs .ucmpcoo chpoea sow mcoHuumem asaHou xwumzamm com-o eo memmec< .NN seemed 85 0—0 (11w 092) aouquosqv 49 0. . “i 0. V N - —- o o’ 1 1 1 1 1 ' I T 1 T '0- ”- '°. “! ". (099V ‘sgs/(louo limo-1) UIGIOJd a 8 '413CK) LZh- A .Gih .3fl- ENMMoni v'O - I-' O . o O O 1Top Figure 23 91 peptides are both dipeptides (Tyr-Arg and Tyr-Hist), and can be separated electroohoretically from other tyrosine-containing tryptic peptides. La- bled C-terminal dipeptides obtained from the purified peptidyl-tRNA fraction of rabbit reticulocyte ribosomes were taken as evidence for the presence of completed globin chains as globyl-tRNA. Peptidyl-tRNA was prepared from ribosomes labeled with tyrosine and subjected to trypsin digestion and high voltage electrophoresis as out- lined under Methods. Authentic samples of the two C-terminal dipeptides were run on either side of the experimental digest. Figure 24 shows the results of this analysis. A small peak of radioactivity was detected in the position of the C-terminal dipeptides. This peak contained approxi- mately 2.5% of the total radioactivity measured. The tryptic digest of tyrosine-labeled peptidyl-tRNA was also ana- lyzed by the peptide mapping technique (see Methods). Figure 25 is a tracing of such a map. The spots staining for tyrosine were cut out and counted, and the distribution of the radioactivity is given in Table VII. Spots 5 and 6 are the two C-terminal dipeptides and contain about 4% of the cpm measured on the map. Chain Separation on C'M-C'eZZuZose The a and 8 chains of rabbit globin can be separated from one an- other on CM-cellulose by the method of Dintzis (1961). 1"C valine-labeled peptidyl-tRNA was brought to 0.1 N in NaOH and incubated at 37° for 2.5 hr to release the peptides from the tRNA. Carrier rabbit globin (20 mg) was added, the sample dialyzed against the starting buffer, and applied to the column. A linear gradient increasing 10-fold in buffer concentration was used to elute the column. The fractions were analyzed for radioactivity 92 . 38:» 8: emcca cm>Hm mew mHHeumu HmucmeHemnxm .emccee HmuHucmuH cm :H muemuceum Hesuw> uHucH mzu ucpgmn ummmmH gown: poem mcpcpeueou-mcwmoexp m :H umuHsmme mHmmeoga -oeuumHm meoemn ummmHu may 0» mmupuamaHu uemvceam uHucmguae mmmuxm eo coHpan< .HuauuemHugm mH ummmwe uHunxem men ease mcpmHee mmuHuamaHu me“ new aHmueem -amm emcee mg» ou umHHaae mcgevceum muHunmnHu mo meoHHHmoa mg» cmmzpma aucmameu -meu msp .muemucmum muHmama new aHeum mvpam me» we memem m>HuHmonim=Hmogau men mpcmmmeame m>oem mewzmeu ms» megz .»HH>HuuaoHuee meg mo comusneemmHu msu mzogm EmemoamH; mg» .aHeum mHQEmm me» we muHm emguHm co :3; mem: pmwzuea» ace me< -exh owucmguam mo mmHaEmm .emucsoo :oHumHHHacpum m cw umucsou use mapeum as m opcm use we: mcmH mHQEmm mg» mo emucpmeme mg» .Amcwguum; mmoeuv meHmoeau so» umcHeum new mcmH mHQEmm mew mo emucmu meg seem ago me: map: as m apepm muHam < .> ooo.~ as e; e\m H eoe we: mesmeeeeoeeemHm . gmH: .eN messed 93 was 3,. cm from Origin Figure 24 94 h . 1.1.1....1...’ -mchoexu me» we AuH>HuumoHume mzh .umcpHpso mew macaw cHgmxgcec mg» .HH> mHaeh cH cm>Hm «H macaw mchHmucoo .vmsoumgummogu macaw m>prmonim=Hmogxu gqu .meoxem: amen: cm>Hm men mmeacmuoea HmucmeH nemaxm .cwaon emHeeeu man «cmwumem Hmaoh .ce:Hou mg“ umuaHm cowumeucmocou meme teem mcwuHexa cH vHomioH mchmmeocH ucmHuoem eemcHH < .coHHmuHHenm mHaEmm squ ummaHm qu>HuumoHume oz .emeezn m>oae men cup: umumeapszam Heu m.n x m.ov :53 iHou mmoHaHHmu-zu e on vaHnnm van .AmcwuHexa z No.o .ueue owegoe z N.ov emeeaa ce=Hoo we mmmcmgu m umchmm um~meHu mHQEmm mg» .umvue we: cHnon HHname umHman -e: me om .eeeeeNPHeeeeee meezOHHee .oem em a: m.~ toe zoez z H.o gee: eeeeeee we; ue_ vamHeaa we mHaeem < .mmoHsHHmu-zu co use ease mmuwuama eo coppecopuumga .eN seemed 99 8 9 o—omw 092) aouoqiosqv O I) N .- 5952 3:09.“. p. 9. on 8 8 8 . -- .--i--fi-<-uhuifl_ié,4 em ee:m_e m . 9.1.0.“. eves-n... on I § 1 § v--4v uudo 3" § i 100 .coHuaHom m.»mem He oH cH umucaou mum: mcopuumea .vHum uHegom um.c gee: espeLeFHeeem use ooH x m.Hv caepee oN-e Hem-eem e as eeeHeee wee Ho: eSH: vaHHmeuamc mm: mH sew mge . 9.d umweeeaa mo mHaEmm He N < .mmuHuamn pcmume: vamnaH-mcwHe> Us" mo :oHumemnmm own; Hmcion .NN messed 101 mm ogaawm DISCUSSION This thesis describes a procedure for the purification of peptidyl- tRNA from rabbit reticulocyte ribosomes. The treatment of ribosomes with concentrated solutions of LiCl has been reported both as a method of pur- ‘3 ifying high molecular weight undegraded rRNA (Barlow et al., 1963), and for purifying ribosomal proteins (Mathias and Williamson, 1964). It has i _ also been reported that tRNA and peptidyl-tRNA are also solubili zed (Bar- , low et al., 1963; Phillips, 1966). Studies reported here demonstrate that the peptidyl-tRNA is quantitatively soluble in 3 M LiCl/4 M urea, and is, by this treatment, cleanly separated from rRNA. During the purification procedure, urea was used to hold the rather insoluble ribosomal proteins in solution until they were removed by the DEAE-cellulose step since peptidyl-tRNA tends to coprecipitate with the ribosomal proteins. The G-200 Sephadex analysis procedure for peptidyl- tRNA depends upon SDS to maintain the ribosomal proteins in solution. The purity of the material obtained by the procedure reported here can be evaluated from both a chemical and an isotopic standpoint. The possible isotopic contaminants are the amino acid used to label the ribo- somes, the corresponding aminoacyl-tRNA, and completed globin chains. All three may be present with the ribosomes used as starting material. Any labeled amino acids are removed during the desalting step on Bio-Gel P-10 (Figure 13) or in the Buffer I wash on DEAE-cellulose (Figure 16). More important for subsequent studies is the possible contamination 102 103 by completed globin chains, for if one wishes to conduct precise analy- ses of the labeling kinetics or patterns of nascent peptide chains, com- pleted and released polypeptide chains, which are also labeled. must necessarily be rigidly excluded. The presence of such labeled chains in- troduces an element of ambiguity into the interpretation of data from such studies. When labeled globin was added to ribosomes and the pepti- dyl-tRNA prepared from them, no radioactivity appeared in the Buffer II or peptidyl-tRNA fraction, demonstrating the ability of the procedure to exclude globin peptides not bound to tRNA (see Figure 17). Soluble glo- bin present in the LiCl soluble fraction is not retained on the DEAE-cel- lulose at this pH and ionic strength, and elutes with Buffer I. The amount of aminoacyl-tRNA present in the purified peptidyl-tRNA is difficult to determine precisely. With material prepared from valine- labeled rabbit reticulocyte ribosomes, it would appear that very little val-tRNA is present. This can be seen in Figure 5 where base hydrolyzed peptidyl-tRNA is analyzed on G-200 Sephadex. Any valine released from tRNA would appear at the end of the elution profile of the G-200 Sephadex column (see Figure 1). No such peak is in evidence. With both the E. coli and rat liver peptidyl-tRNA. however, base hydrolysis released labeled material to the amino acid position of the G-200 Sephadex column (Figures 20C and 21C). It should be noted that a mixture of amino acids was used to label the E. coli and rat liver ribosomes and therefore the presence of any of the species of aminoacyl-tRNA would be detected by this method of analysis. Approximately 25% of the radioactivity in the purified E. coli peptidyl-tRNA appeared to be in aminoacyl-tRNA and 75% in pepti- dyl-tRNA. These data for the rat liver peptidyl-tRNA preparation are 5% and 95% respectively. 104 w The bulk of the chemical contaminants to be removed by this puri- fication scheme consists of rRNA and ribosomal protein. The LiCl step removed all the rRNA as Figure 12 demonstrates. Ribosomal proteins are even more basic than globin and would be expected to pass through the DEAE-cellulose column in the Buffer I elution. Analysis of the fractions of Figure 17 for their protein content confirmed this expectation. In addition, measurement of the protein concentration of a preparation of purified peptidyl-tRNA from 40 mg of ribosomes showed no detectable poly- peptide material. This is the expected result if one assumes that only about 0.1% of the mass of the ribosome is nascent peptide chains. Thus, 40 mg of ribosomes would yield less than 40 ug of protein in the peptidyl- tRNA, and this amount, present in the tinal volume of the preparation, is below the limits of detection by the Lowry method. Uncharged tRNA would be expected to be present in the purified peptidyl-tRNA preparation. Initial purification attempts sought to use the pooled peptidyl- tRNA peak from the G-200 column (Figure 1). However, complete removal of SDS from the resulting material could not be accomplished and the traces of the detergent present interfered with subsequent attempts at ion ex- change chromatography of the peptidyl-tRNA. When SDS was removed by di- alysis against dilute aqueous solutions, the ribosomal proteins precipi- tated, and the peptidyl-tRNA was precipitated with them. Other workers have reported the use of phenol extraction to purify polylysyl-tRNA from bacterial ribosomes programmed by poly A (Rychlik, 1966; Bretscher et al., 1965; Gottesman, 1967). Attempts to apply phenol extraction to the preparation of peptidyl-tRNA from rabbit reticulocyte ribosomes were unsuccessful in that most of the peptidyl-tRNA precipitated with the protein into the phenol phase. In addition, salt washing and n1 105 dissociation of the ribosomes into subunits by dialysis against EDTA or treatment with polyvinyl sulfate were unsuccessful iniissociating the peptidyl-tRNA from the large and small ribosomal subunits. A signi- ficant proportion of the nascent chains are not released from the ribo- some upon dissociation into subunits, and Philipps (1966) has suggested that this bound fraction represents the longer nascent chains. The studies reported here with rat liver and E. coli ribosomes would indicate that these methods are of general use in preparing peptidyl-tRNA from other ribosomal sources. Attempts to purify polyphenylalanyl-tRNA from E. coli ribosomes programmed with poly U gave poor yields and the procedure reported here may not be the method of choice with ribosomes programmed by certain synthetic mRNA's. The results shown in Figure 5 warrant some discussion in that the peptides released from peptidyl-tRNA by treatment with base appear rather more uniform in size than would be expected for a random population of nascent globin chains. This phenomenon apparently results from the pres- ence of SDS in the elution buffer. This anomaly deserves mention because of the widespread use of $05 as a dissociating agent. SDS seems to bind to the peptides which then behave on the G-200 Sephadex column as an SDS-peptide complex with very little apparent heterogeneity. For example, a tryptic digest of the purified peptidyl-tRNA containing labeled tryptic peptides with molecular weights from a few hundred to about three thou- sand (Figure 68), is almost indistinguishable on the G-200 Sephadex column from the nascent chains in Figure 5 which would be expected to have a molecular weight range from several hundred to sixteen thousand. When the dipeptides Tyr-Arg and Tyr-Hist were analyzed on the G—200 Sephadex column, they eluted before the inclusion volume of the column and were 106 resolved from one another although their molecular weights differ only by 18. Arginine, a positively charged amino acid, eluted not in the position indicated for amino acids in Figure 1, but nearer the tRNA marker. Thus, a good deal of the binding of the SDS is no doubt elec- trostatic. In spite of these difficulties, peptidyl-tRNA and peptides released from tRNA were clearly distinguished from one another. Detailed studies of the size distribution of nascent peptides will have to be pur- sued using a different analytical method. The finding that completed globin chains are present on ribosomes as peptidyl-tRNA may be important to the understanding of the overall process of hemolgobin biosynthesis. Previous workers have reported the presence of completed globin chains on ribosomes (Hunt et al.. 1968a; Baglioni and Campana, 1967). Hunt et al. (1968a) suggest that these complete chains represent contamination of the ribosomes by soluble globin. Baglioni and Campana (1967) have reported that completed a chains found on ribosomes are not bound to tRNA but represent an import- ant ribosome-bound intermediate in globin assembly. The studies reported here indicate that a number of completed globin chains are present on ribosomes as peptidyl-tRNA. The work of Hunt et at. (1968) on the distribution of ribosomes along the hemoglobin mRNA indicates a random distribution. This implies the presence of equal numbers of all possible peptide lengths between valyl-tRNA (the N-terminal amino acid of both a and B globin chains) and completed a and B globyl-tRNA. If this is correct, one should find a weighted average between 1/141 and 1/146* completed chains on the *The a and B chains contain 141 and 146 amino acids respectively, but there is reported to be about 10% more nascent a than B chains (Hunt 8t aZ., 19583). 107 ribosome. Using these assumptions, one can calculate.the theoretical percentage of cpm in the C-terminal tryptic dipeptides of nascent chains, when Tyr is used to label ribosomes, as 0.6%. The observed value with ribosomes labeled in a cell-free system, was greater than 2% of the total cpm present in the C-terminal peptides. There are several possi- ble explanations. First, these dipeptides might arise from a non-globin protein made in the cell-free system. Such an explanation appears high- ly unlikely since more than 90% of the protein made in reticulocytes is hemoglobin. Second, the distribution of nascent chains on ribosomes incubated in the cell-free system may be significantly different than that which occurs in vivo. 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