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A , - . :3 'IO‘HV5' _, 3 I'. ‘ . »‘a 'u 1,!” 333.36%:‘333L3: ,,,,f,,,33:3l3 7‘, . : , . lawn-47533 333 ‘ 3 n ., 3 3 . 3 W3 333.3333 “ 3'» 3' .‘IP‘ 3, .' 33 33 1.," 33,333333333333333 ‘ ‘3.',' 3", ,3, #3333333“ ., 33 333 I I ' 3 3"! ,,,,‘,,333, , 333333'M ' I ,“f “I, 3 3333313333 '3 33-333 ,3, ,, 3, ,,,l,3v,',‘, 3, 3333333333 3333? ”33“!" 3333; (3.3333 3333333133331 333 ’3“; 333,3 .3, l' 3,, 333,, u, .3 , , , , 3,3,33 33333'3 . , '53,», ' , 3 I‘ll“ 3, I 3, '3‘ 3"3'3'3 3333'- "'u'3"u"13°.‘. .313 33 ”u '331 1l3nL3lm3333n333|IIIHHMMhLiM icyML 33333333 THESIS This is to certify that the thesis entitled The Relationship Between Messenger RNA and Nascent Peptide Size Distribution: The Role of Messenger RNA Integrity presented by Howard Paul Hershey has been accepted towards fulfillment of the requirements for Ph.D. Jegeem Biochemistry flea/XML ajor professor Date Wk?! /7// 0-7539 MSU LIBRARIES a! RETURNING MATERIALS: Place in bodk drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. THE RELATIONSHIP BETWEEN MESSENGER RNA AND NASCENT PEPTIDE SIZE DISTRIBUTION: THE ROLE OF MESSENGER RNA INTEGRITY By Howard Paul Hershey A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1981 ABSTRACT THE RELATIONSHIP BETWEEN MESSENGER RNA AND NASCENT PEPTIDE SIZE DISTRIBUTION: THE ROLE OF MESSENGER RNA INTEGRITY BY Howard Paul Hershey An investigation into the origion of the nonuniform rabbit globin nascent peptide sizes observed during translation of globin mRNA in different mRNA dependent cell-free protein synthesizing systems was performed. Accumulations of discrete size nascent peptides similar to those seen during globin synthesis in reticulocyte lysates were observed during translation of globin mRNA in the wheat germ cell-free protein synthesizing system. Globin mRNA translated in the mRNA dependent reticulocyte lysate gave rise to a distorted nascent peptide size distri- bution. Analysis of the mRNA dependent reticulocyte lysate for the presence of nascent peptides in the absence of added mRNA showed that a reproduc- ible size distribution of nascent peptides was present in this protein synthesizing system. These nascent peptides were postulated to arise from initiation of protein synthesis on globin mRNA fragments remaining in the mRNA dependent lysate following micrococcal nuclease digestion. Pactamycin inhibited the initiation of protein synthesis on these frag- ments and revealed an apparent inability to terminate protein synthesis normally on mRNA fragments. No nascent peptides were found in the wheat germ cell-free protein synthesizing system in the absence of added mRNA. Pactamycin treatment Howard Paul Hershey of the globin mRNA directed wheat germ cell-free system removed the nascent peptide population from polysomes synthesizing globin. Transla- tion of globin mRNA fragmented by $1 nuclease gave rise to a population of nascent peptides which remained following pactamycin addition to the cell-free protein synthesizing system. Analysis of 3' and labeled globin mRNA showed that fragments were not present in the globin mRNA preparations used to direct globin synthesis in the wheat germ cell-free protein synthesizing system. Translation of 3' end labeled globin mRNA demonstrated that no fragments of globin mRNA were generated during protein synthesis in this system. The data in this thesis document the presence of globin mRNA frag- ments capable of normal initiation, but not termination of protein synthesis by the normal sequence of events. Data are also presented showing that nascent peptides observed during translation of globin mRNA in the wheat germ cell-free protein synthesizing system are not due to globin mRNA fragments present in the protein synthesizing system. ACKNOWLEDGEMENTS I would like to thank Drs. H. J. Kung, A. Revzin, R. Patterson, and H. Hells for serving on my graduate advisory committee. Special thanks are given to Dr. Allan J. Morris for his guidance, help, patience, and good humor during my graduate studies. ii TABLE OF CONTENTS LIST OF TABLES. ........ . . ...... . . . . . . . . . . LIST OF FIGURES O ....... 0 O O O O 0 O O O O O 0 O O O O O 0 INTRODUCTION. 0 O O O O O 000000 O O O O O O O O O O O O O O 0 Protein Biosynthesis . . . . . . . . . . . . . . . . . . . . . G]0b1. n Bi osyntheSi S O O O O O 0 O O O O O O O O O O O O O O O Pr‘mary Sthture Of RNA 0 I I I O O O O O O O O O 0 0 O O O 0 RNA Secondary Structure. . . . . . . . . . . . . Secondary Structure and RNA Function . . . . . . . . . . . . . Eukaryotic mRNA Structure and Function . . . . . . . . ..... Structural Features of Globin mRNA . . . . . . . . . . . . . . Nascent Globin Peptide Nonuniformity . . . . . . . . . . . . MATERIALS 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 METHODS O O O O O O O O O O O O O O I O O O O O O ..... O O O 0 Preparation of Rabbit Reticulocytes. . . . . . . . . . . . . . . Preparation of the Reticulocyte Lysate . . . . . . . . . . . . . Preparation of the Wheat Germ Cell- Free Protein Synthesizing System Determination of the Rate of mRNA Directed Protein Synthesis in the Wheat Germ Cell-Free System. . . . . . . . . . . . . . Preparation of the mRNA Dependent Reticulocyte Lysate Cell-Free Protein Synthesizing System . . . . . . . . . . . . . . . . . Preparation of Rabbit Globin mRNA. . . . . . . . . . . . . . . . Identification of Products from Cell-Free Protein Biosynthesis: Separation of a- and a-Globin . . . . . . . . . . . . . . . . Polysome Labeling for Preparation of Peptidyl-tRNA . . . . . . . Peptidyl-tRNA Purification . . . . . . . . . . . . . . . . . . . Preparation of the Bio-Gel A 0.5m Column and Analysis of Nascent Peptide Size Distribution . . . . . . . . . . . . . . . . . . Cyanogen Bromide Cleavage of a- and s-Globin . . . . . . Determination of Distribution Coefficients . . . . . . . Recrystallization of Guanidinium Chloride. . . . . . . Preparation of Stock Urea, Buffer I, Buffer II, and DESZ Separation of a- and B-Globin mRNAs. . . . . . . . . . . Translational Purity of Rabbit a- and s-Globin mRNA. . . Limited Nuclease $1 Digestion of Globin mRNA . . . . . . iii 3' End Labeling of Globin mRNA . . . . . . . . . . . . . . . . 5' End Labeling of Globin mRNA . . . . . . . . . . . . . . . . RESULTS 0 O O O O O O O O O O O O O I O O O O O O I O O O O O O 0 Wheat Germ Cell- Free System. . . . . . . . . . . . . . . . . Purification of Rabbit Globin mRNA . . . . . . . . . . . . . . Characterization of the Products Synthesized in Response to Added Globin mRNA . . . . . . . . . . . . . . . . . . . . Calibration of the Bio-Gel A 0.5m Column . . . . . . . . . . Methylmercuric Hydroxide Treatment of Globin mRNA. . . . . . Limited Digestion of Globin mRNA with $1 Nuclease. . . . . . Effects of $1 Digestion of Globin mRNA on the Nascent Chain Size Distribution . . . . . . . . . . . . . . . . . . . . . Preparation of a mRNA Dependent Reticulocyte Lysate. . . . . . Destruction of Endogenous mRNA in the Reticulocyte Lysate. . . Nascent Peptide Analysis from the mRNA Directed Synthesis of Globin in the mRNA Dependent Reticulocyte Lysate. . . . . . Analysis of Background Nascent Peptides in the mRNA Dependent Reticulocyte Lysate in the Absence of Added mRNA. . . . . . . Effect of Micrococcal Nuclease Digestion of Endogenous Globin mRNA on the Nascent Peptide Size Distribution . . . . . . . Confirmation of Background Nascent Peptides in a Commercially Available mRNA Dependent Reticulocyte Lysate. . . . . . . . Effect of Pactamycin on the Background Nascent Peptide Size Distribution in the mRNA Dependent Reticulocyte Lysate. . . Analysis for Background Nascent Peptides in the Wheat Germ System. . . . . . . . . . . . . . . . . . . . . . . . . . . The Effect of Pactamycin on the Nascent Peptide Size Distribuion in the Wheat Germ Cell- Free System. . . . . . . . . . . . . Separation of a— and s-Globin mRNA . . . . . . . . . . . . . . Assessment of the Translational Purity of a- and B-Globin mRNA Determination of the a- and B-Globin Nascent Peptide Size Distribution. . . . . . . . . . . . . . . . . . . . . . . . Comparison of the a- and s-Globin Nascent Chain Size Distributions . . . . . . . . . . . . . . . . . . . . . . . The Elution Profile of Nascent Peptides Resulting from the Translation of a Recombined Mixture of Purified a- and B- Globin mRNA . . . . . . . . . . . . . . . . . . . . . . . . 5' End Labeling of Globin mRNA . . . . . . . . . . . . . . . . 3' End Labeling and Translation of Globin mRNA . . . . . . . . DISCUSSION. 0 O O O O O I O O O O O O O O O I O O O O O O O O O 0 LIST OF REFERENCES. 0 O O O O O O O O O O C O O O O O O O O O O O 0 iv Page 106 111 121 127 I34 139 142 145 I48 154 173 Table II III LIST OF TABLES Final Concentrations of the Components of the Reticulocyte Lysate Cell-Free System . . . . . . . . . . . 20 Final Concentrations of Components Added to the Wheat Germ Cell-Free System. . . . . . . . . . . . . . . . 23 Final Concentrations of Components in the 3' Terminal Labeling of Globin mRNA . ....... . . . . . 34 10. 11. LIST OF FIGURES Page The incorporation of [3H]-leucine into protein as a function of time by the wheat germ cell-free system in response to added globin mRNA. . . . . . . . . . . . . . . 38 Protein synthesis as a function of Mg(0Ac)2 and KOAc concentrations in the wheat germ cell-free system. . . . . 41 The effect of mRNA concentration on the rate of protein synthesis in the wheat germ cell-free system . . . . . . . 43 The rate of protein synthesis in the wheat germ cell-free system in response to globin mRNA prepared by different mEthOdS I I I I I I I I I I I I I I I I I I I I I I I I I I 45 Analysis of the soluble products synthesized by the wheat germ cell-free system in response to globin mRNA by CM cellulose column chromatography. . . . . . . . . . . . . . 47 The analysis of the a- and e-globin synthesized by the wheat germ cell-free system in response to globin mRNA by 81 O'GEI A 005'“ 9e] filtrationI I I I I I I I I I I I I I I 50 Analysis of the size distribution of [3H]-tryptophan labeled nascent peptides from the mRNA directed wheat germ ce1l-free sySteI“ I I I I I I I I I I I I I I I I I I I I I 52 Calibration of the Bio-Gel A 0.5m analytical gel filtration c01umn I I I I I I I I I I I I I I I I I I I I I I I I I I 55 The effect of methylmercuric hydroxide denaturation of globin mRNA on the rate of protein biosynthesis in two different cell-free protein synthesizing systems . . . . . 58 The effect of methylmercuric hydroxide denaturation of globin mRNA on the size distribution of nascent peptides in the mRNA directed wheat germ cell-free system . . . . . 61 The rate of protein synthesis in the wheat germ cell-free system using globin mRNA subjected to nuclease digestion . 64 vi Figure 12. 13. I4. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. The effect of limited $1 nuclease digestion of globin mRNA on the nascent peptide size distribution in the globin mRNA directed wheat germ cell—free system . . . . . The size distribution of nascent peptides in the globin mRNA directed wheat germ cell-free system in the absence Of 51 nuc‘ease I I I I I I I I I I I I I I I I I I I I I I The effect of the presence of inactive micrococcal nuclease in the reticulocyte lysate on the elution profile of globin nascent peptides . . . . . . . . . . . . The time course of micrococcal nuclease inactivation of globin mRNA in the reticulocyte lysate . . . . . . . . . . The globin mRNA directed incorporation of [3H]-leucine into protein in the mRNA dependent reticulocyte lysate . . The elution profile of [3HJ-tryptophan labeled nascent peptides from the globin mRNA directed mRNA dependent reticulocyte lysate. . . . . . . . . . . . . . . . . . . . Analysis of the soluble products synthesized by the globin mRNA directed microccocal nuclease inactivated reticulocyte lysate by CM cellulose colunn chromatography . . . . . . . The elution profile of nascent peptides from the mRNA dependent reticulocyte lysate in the absence of added mRNA I I I I I I I I I I I I I I I I I I I I I I I I I I I The effect of the time of micrococcal nuclease digestion of the endogenous mRNA of the reticulocyte lysate on the size distribution of nascent peptides produced . . . . . . The rate of globin mRNA directed protein synthesis in a commercially available mNRA dependent reticulocyte lysate I I I I I I I I I I I I I I I I I I I I I I I I I I The elution profile of [3HJ-leucine labeled nascent peptides from the globin mRNA directed commercial mRNA dependent reticulocyte lysate. . . . . . . . . . . . . . . The size distribution of nascent peptides of the commercial mRNA dependent reticulocyte lysate in the absence of added mRNA I I I I I I I I I I I I I I I I I I I I I I I I I I I The effect of pactamycin on protein synthesis in the mRNA directed mRNA dependent reticulocyte lysate. . . . . . . . The elution profile of nascent peptides of the mRNA dependent reticulocyte lysate prior to pactamycin addition I I I I I I I I I I I I I I I I I I I I I I I I I vii 67 69 73 76 78 81 83 86 9O 94 96 98 101 103 Figure 26. 27. 28. 29. 30. 31. 32. 33. 34. 35I 36I 37. 38I 39. The elution profile of nascent peptides from the mRNA dependent lysate following a 10 minute pactamycin ChaseI I I I I I I I I I I I I I I I I I I I I I I I I I I The elution profile of nascent pe tides from the mRNA dependent lysate following a 25 m nute pactamycin ChaseI I I I I I I I I I I I I I I I I I I I I I I I I I I The effect of excess micrococcal nuclease on the nascent peptide size distribution of the mRNA dependent lysate . . The elution profile of nascent peptides from the wheat germ cell-free system in the absence of added mRNA . . . . The effect of pactamycin on the rate of protein synthesis of the globin mRNA directed wheat germ cell-free system. . The nascent peptides of the globin mRNA directed wheat germ cell-free system before pactamycin addition . . . . . The elution profile of the nascent peptides of the globin mRNA directed wheat germ cell-free system 20 minutes after pactamycin addition. . . . . . . . . . . . . . . . . The size distribution of nascent peptides remaining after a 20 minute pactamycin chase in the wheat germ cell-free system programmed with 10 minute 51 nuclease digested globin mRNA. . . . . . . . . . . . . . . . . . . . . . . . The slicing patterns used to recover mRNA fractions from preparative polyacrylamide gels. . . . . . . . . . . . . . Gel electrophoresis of the globin mRNA fractions after preparative electrophoresis. . . . . . . . . . . . . . . . Analysis of the [3HJ-isoleucine labeled soluble products from the a-globin mRNA directed wheat germ cell-free system by CM cellulose column chromatography . . . . . . . Analysis of the [3H]-tryptophan labeled soluble products from the a-globin mRNA directed wheat germ cell—free syStan I I I I I I I I I I I I I I I I I I I I I I I I I I The size distribution of the [3H]-isoleucine labeled nascent peptides from the a-globin mRNA directed wheat gem cell-free 5yStall. o o o o o e o o o o o o o o o o o o The size distribution of the [3HJ-tryptophan labeled nascent peptides from the a-globin mRNA directed wheat genIICEII-fY'EESYStem.ooo........o.oo... viii Page 104 108 110 113 115 118 120 123 126 129 131 133 136 138 Figure Page 40. The size distribution of the [3H]-tryptophan labeled nascent peptides from the a-globin mRNA directed wheat gem cell-free syStano I I I I I I I I I I I I I I I I I I 141 41. The size distribution of nascent peptides from the translation of a mixture of purified a- and s-globin mRNAs in the wheat germ cell-free system . . . . . . . . . 144 42. Autoradiogram of the separation of the 5' end labeled products of globin mRNA by polyacrylamide gel electrophoresis. . . . . . . . . . . . . . . . . . . . . . 147 43. Autoradiogram of the separation of 3' end labeled globin mRNA by polyacrylamide gel electrophoresis . . . . . . . . 150 44. Autoradiogram of the separation of 3' end labeled globin mRNA by polyacrylamide gel electrophoresis after translation in the wheat germ cell-free system . . . . . . 153 ix INTRODUCTION Protein Biosynthesis Messenger RNA (mRNA) contains the necessary information within its nucleotide sequence to specify a polypeptide sequence. Ribosomes read the coding sequence of the mRNA from the 5' end to the 3' end (1), poly- merizing amino acids sequentially in a stepwise fashion. Polypeptides are synthesized from the N terminal end to the C terminal end of the protein (2,3). Each ribosome traversing the mRNA contains a single nascent chain that is growing at the rate of one amino acid per triplet codon encountered by the ribosome (4). The aminoacyl-tRNA molecules read each codon in the mRNA sequence and the growing polypeptide is transfer- red to the new tRNA. The rate of protein biosynthesis may be regulated by any of the steps involved in protein synthesis (initiation, elongation and termina- tion steps) or by the amount of mRNA available for the synthesis of a given protein. The level of a given protein in a cell therefore is governed by a balance between the stability of each mRNA in the cyto- plasm, the rate at which protein is being made from that mRNA, and the rate at which the protein product is being degraded (5,6,7). Globin Biosynthesis The synthesis of a— and a-globin in mammalian reticulocytes has been extensively studied. Mammalian reticulocytes are non-nucleated cells that are synthesizing a- and a-globin from residual mRNA and ribosomes. These polysomes will soon be eliminated along with the remaining mito- chondria in the cell, marking the transition of the reticulocyte to the mature erythrocyte. Greater than 95% of the protein synthesis in the reticulocyte is dedicated to the production of a- and B-globin (8). Alpha globin is produced in a slight excess in rabbit reticulocytes (9). The observed translation rate of a- and s-globin are both equal, each protein requir- ing 200 seconds for completion (10,11). The size of polysomes involved in B-globin synthesis has been shown to be larger than the observed size of polysomes synthesizing a-globin (12). Lodish found a—globin mRNA to be present in 40% greater quantities than B-globin mRNA in the rabbit reticulocyte (11). Lodish also found that the initiation of protein synthesis by ribosomes on e-globin mRNA was more efficient than the initiation rate observed for the a-globin mRNA. The rate of initiation observed for a-globin mRNA was 65% as fre- quent as that observed for B-globin messenger RNA. This lowered initia- tion rate for a-globin synthesis coupled with the approximate 40% excess of a—globin mRNA as compared with a-globin mRNA in the reticulocyte gives a final ratio of a- to B-globin synthesis of approximately 1:1. This faster initiation rate for B-globin mRNA was also proposed to account for the increased polysome size observed during s-globin synthesis. Primary Structure of RNA Ribonucleic acids are linear polymers of nucleoside monophosphates. Naturally occurring RNA is synthesized from the four ribonucleotide tri- phosphate by joining of the corresponding nucleoside monophosphates through 3'-—-95' phosphodiester bonds. In addition to the four major bases found in RNA, some minor bases are also found in RNA as are some modifications of the ribose moiety (such as 2'-0-methyl derivatives). The primary structure of RNA may contain obviously available information as is the case with mRNA where the coding sequence specifies the order of amino acids for a specific protein. The information in RNA may also be expressed in a more subtle manner. The order of arrangement of bases in a polyribonucleotide also defines a series of intramolecular structural features which result in a conformation that the molecule may adopt. A knowledge of conformations adopted by different RNA molecules may well be necessary before an understanding of the functional aspects of these molecules can be accomplished (14). RNA Secondary Structure Elucidation of the secondary structure of large polyribonucleotides is an essential step in the understanding of RNA function. Secondary structure components in RNA species would be expected to be different for each RNA species analyzed due to the different nucleotide sequence present in each RNA. Initial studies involving elucidation of RNA secondary structure were based on the known hypochromic shift that occurs when a polynucleo- tide adopts a more helical conformation from a more random conformation. This shift to lower absorbance is due primarily to base stacking inter- actions which help in the stabilization of the helices formed. The use of thermal denaturation to disrupt regions of helical structure has been widely used to determine the extent of secondary structure in RNA. The hyperchromic shift resulting from heat denaturation of RNA has been used to estimate the helical content of total ribosomal RNA (15), 165 and 185 ritrosomal RNA (16), ovalbumin mRNA (17), globin mRNA (18) and 5S RNA (19). Using the method of hypochromic shift measurement following thermal denaturation estimates of the helical content within RNA have ranged from 55 to 75%. The method, however, does not allow prediction of the location of the regions of putative helical structure. The decrease in susceptibility of regions involved in base pairing to nuclease degradation has also been used to study RNA secondary structure. Digestion of 165 and 185 rRNA with ribonuclease T1 and T2 revealed regions of these molecules resistant to these structure specific nucleases (20). The resistance of 16S RNA to ribonuclease T1 action lead Ricard and Sales to predict long regions of base paired structures (21). Fiers gt 31. used limited ribonuclease T1 digestion to generate defined length fragments of the MS2 coat protein genome (22). Later work by Fiers gt a1. employed the single strand specificity of ribonuclease T1 to complete the determination of the base sequencing the entire M32 genome (23). The sensitivity of certain regions to digestion with nuclease in conjunction with prediction of secondary structure by theoretical calculations of free energies of proposed loops was used to predict the secondary structure map of the complete M52 genome. The secondary structure of the potato spindle tuber virion has also been predicted by the use of sequence data to predict regions of secondary structure supplemented with nuclease sensitivity data (24). $1 nuclease, a single strand specific endonuclease, has been extensively used to map regions of secondary structure within RNA mole- cules. The digestion products resulting from $1 digestion of 16S ribosomal RNA have been used to determine the interacting sequences within the molecule (25). Flashner and Vournakis subjected globin mRNA to limited digestion with $1 nuclease under non-denaturing conditions and found that up to 75% of the globin mRNA sequences are resistant to the action of the nuclease (26). The data from these experiments with globin mRNA were interpreted to support a structural model of globin mRNA in which a high degree of helical structure was present within the primary sequence. Wurst gt 31. have developed a method which localizes regions of secondary structure within an RNA sequence up to 150 nucleotides long. The data obtained from the specific cleavage of 5' [32PJ-labeled RNA using base specific nucleases in conjunction with analysis of the fragments resulting from RNA limited $1 cleavage of the labeled RNA allows accurate prediction of secondary structure (27). This method was used to correctly predict the secondary structure of two different transfer RNA molecules whose secondary structures were previously known. Pavlakis £5 31. used a modification of the method of Wurst to predict the secondary structure of the 5' end of both the a- and s-globin mRNAs (28). Branlant gt 31. have developed a method using Naja oxiana nuclease in the presence of high concentrations of magnesium to map secondary structure in RNA (29). The Naja oxiana nuclease under conditions of high magnesium concentration degrades regions of RNA-RNA interaction specifically. The method has been used to determine the secondary structure of U4 nuclear RNA. The RNA was labeled on the 3' end with 32F and partially digested with T1 and $1 nucleases to ascertain the locations of single stranded regions of the RNA. Naja oxiana nuclease was then used to cleave the RNA specifically at the base paired regions. The digestion products were fractionated on polyacrylamide gels and identified by comparison with ribonuclease T1 digestions performed under denaturing conditions along with alkali digested RNA data. The secondary structure data was then used to assemble a secondary structure map of U4 RNA (30). The recent advent of base sequencing methods for both DNA (31,32,33) and RNA (34,35) is increasing the nunber of RNA molecules for which a primary sequence is known. The primary sequence of an RNA can be used to generate a map of secondary structure within the nucleotide sequence. The relative stabilities pr0posed structures can be analyzed by procedures developed by Tinoco gt 31. (33,34). These procedures predict the free energy of formation of possible helical structures arising from a known primary sequence using a set of experimentally developed parameters. Another method of predicting base pairing has been develOped by Salser (38). The method calculates the most stable combinations of compatable regions of a long RNA sequence by calculations of the free energy of formation for a given sequence of bases. Pavlakis gt 11. (28) have pointed out that proposed secondary structure models generated by computer using only primary sequence data do not always fully agree with structure maps assembled from structure specific nuclease cleavages. Future methods for predicting RNA secondary structure by computer analysis of sequence data may soon include secondary structure data from nuclease mapping studies along with primary sequence data to generate more accurate secondary structure maps. Secondary Structure and RNA Function Lodish was among the first investigators to observe a relationship between RNA structure and function (39). By denaturation of the structure of the bacteriophage f2 genome using formaldehyde, Lodish was able to increase the rate of f2 RNA directed protein synthesis by up to 20 fold for selected proteins coded for in the f2 genome. In addition, several protein products were synthesized that did not correspond to any known authentic f2 gene products. These results were interpreted to demonstrate the importance of the secondary structure of f2 RNA to its proper function. Translation of the replicase gene in M52 bacteriophage is known to require synthesis of at least the N-terminal half of the coat protein cistron (40,41). The translation of the N-terminal half of the coat protein gene has been postulated to alter the conformation of the M52 genome. This change exposes the replicase initiator which is proposed to be based paired to the 5' half of the coat protein cistron in the absence of translation. (42). Ahlquist gt g1. have studied the 3' terminal secondary structure in a variety of viruses of the bromovirus family (43). Their results showed that the secondary structures generated for the 3' ends of all three RNA classes have been retained among all the bromoviruses. The structures generated not only showed conservation of the general shape of the 3' region, but also conservation of specific sequences within these regions. These results were confirmed by ribonuclease T1 digestions. This conservation of the 3' end was postulated to be necessary for retaining the ability to be aminoacylated lg 1139. Recently, structural analysis of the ribosome binding sites for three bacteriophage T7 class 111 genes was performed by Rosa (44). All three binding sites contained a sequence of bases complimentary to the 3' end of 165 RNA, in keeping with the hypothesis of Shine and Dalgarno (45). The three binding sites studied appeared to have initiator regions which were buried in secondary structure when analyzed by the method of Wurst gt 31. (27). The secondary structure proposed for these binding 8 sites was postulated to aid in the binding of ribosomes to the correct initiation site for protein synthesis, and possibly protect the 5' end of the transcript from exonucleases attack. Robertson gt 91. studied the binding of ribosomes to 1251- labeled globin mRNA (46). Following nuclease digestion of the globin mRNA-initiator complex with pancreatic ribonuclease these investigators found a set of specific fragments of globin mRNA which were protected by ribosome binding. The nucleotide sequences of the fragments were then determined. The results showed that binding of the 405 ribosomal subunit to globin mRNA protected an 80 base sequence of the message, while assembly of the complete ribosome protected only half as much of the same region. These data suggested a conformational change in the globin message structure during assembly of the 805 initiation complex. Eukaryotic mRNA Structure and Function Messenger RNA can be subdivided structurally into four distinct regions of primary structure. These are a 5' untranslated region, a coding region, a 3' non-translated region and a Poly(A) region which is present on most but not all eukaryotic messages. The 5' untranslated region consists of the base sequence from the 5' end of the mRNA to the initiator codon. The 5' end of this region usual- ly begins with one of the three cap structures (see 47 for review). Messenger RNA molecules that have been uncapped have been shown to display a greatly reduced template activity when translated in the wheat germ cell-free system (48). In addition, 7-methyl guanylic acid, a cap analog, inhibits the translation of capped, but not uncapped mRNA (49). These data led to the suggestion that the cap may function by stimulation of the rate of initiation of protein synthesis (50). Beyond the cap is the 5' untranslated region or leader sequence which continues to the initiator codon. This region has been observed to be variable in length in the mRNAs thus far sequenced. The suggestion has been made that a nucleotide sequence in the 5' non-coding region may serve as a binding site for the 3' terminus of the 18S RNA in the 405 ribosomal subunit. This would be analogous to the situation put forth for mRNA and 165 rRNA interactions during initiation of protein synthesis in procaryotes by Shine and Dalgarno (45). This hypothesis usually correlates well with the observed interaction of 16S RNA of prokaryotic ribosomes with the initiator region in prokaryotic mRNA. The experimental evidence does not appear to support a similar type of interaction for eukaryotic mRNA. Studies of the 5' noncoding region of eukaryotic mRNA thus far sequenced have not generally shown a sequence capable of base pairing with 185 RNA and initiation of protein synthesis may proceed by a mechanism that is not analogous to that seen in prokaryotes (51). The coding region of mRNA consists of the sequence of bases that specifies the amino acid sequence of the protein to be biosynthesized. Interestingly, specific mRNA molecules display a non-random use of triplets when the degeneracy of the genetic code provides a choice. This preference for the use of specific triplets in a series of synonomous codons seems to be unique for each specific mRNA since no patterns emerge when the choice of codons is compared among different mRNA. Beta globin mRNA shows a weak preference for the base G in the third position of codons while u—globin mRNA shows a marked preference for codons ending in C (52,53). Codon choices used most frequently in the bacteriophage M52 RNA favor those codons that end in U. While the significance of this bias for specific codons remains to be determined, some investigators 10 have suggested that the non-random use of codons represent the selection of a specific codon within a synonomous pool such that secondary structure within the mRNA can be optimized (54). Following the coding region of the mRNA is the 3' untranslated region. This region continues to the start of the poly(A). The length of this 3' region varies considerably among different mRNAs whose nucleotide sequences are known. The function of the 3' untranslated region is not yet understood. Studies on the sequences of the duplicated human a- and a-globin genes provide few clues to the function of this region. These genes are thought to have arisen from a gene duplication and have developed little heterogeneity between the duplicated genes over time with the exception of the 3' non-coding regions which are unexpectedly divergent (55). In the Bromoviruses, however, the sequence homology of the 3' untranslated regions of the three classes of RNA found in the virions has been highly conserved (43). Proposed secondary structures of these viral RNA molecules also show a striking similarity indicating the sequence homology may have been retained so that secondary structure could be conserved. The poly(A) region which follows the 3' untranslated region is present in approximately 70% of all eukaryotic mRNA (56). The poly(A) at one time had been postulated to function in the transport of mRNA from the nucleus to the cytoplasm, however, the transport and translation of non-poly(A) containing mRNA molecules has since been shown (57,58). The proposal has also been made that the poly(A) may function in the deter- mination of the functional half-life of mRNA. Deadenylated globin mRNA has been shown to display no alteration in the initial rate of protein synthesis in cell-free protein synthesizing systems when compared with 11 poly(A) containing globin mRNA (59). At longer incubation times, how- ever, the template ability of the deadenylated globin mRNA was lost more rapidly than the rate of loss observed for adenylated mRNA (59,60). The stability of the deadenylated globin mRNA could be restored by readenylation of the messenger RNA (61). Although it seems that poly(A) enhances mRNA stability in 11359, a defined function on this region has yet to be documented. Structural Features of Globin mRNA Globin mRNA is known to undergo an increase in absorbance at 260 nm following thermal denaturation. This increase in absorbance was shown to be more than could be accounted for by a random c0polymer structure. Further studies on the thermal deaturation of globin mRNA by Holder and Lingrel indicated that up to 68% of the bases in the mRNA could be found involved in secondary structure (18). Purified globin mRNA has also been subjected to nuclease digestion with $1 nuclease. Flashner gt 31. subjected globin mRNA to digestion with $1 nuclease under conditions where secondary structure would be stabilized (26). They found up to 75% of the globin sequence was resist- ant to the single strand specific nuclease. The $1 digestion products of globin mRNA were found to be oligonucleotides ranging in size up to 80 nucleotides in length. Pretreatment of globin mRNA by methods known to disrupt helical structures such as heat or formaldehyde treatment greatly increased the $1 susceptibility of the mRNA, while high salt concen- trations lowered nuclease susceptibility. From these data and the previous work of Vournakis gt g1. (62), globin mRNAs were postulated to contain an extensive degree of helical structures within the a- and B-globin mRNA molecules. 12 Dubochet gt g1. (63) used dark field electron microscooy to study the structure of both duck and rabbit globin mRNA and globin messenger ribonucleoprotein particles. Following staining with uranyl acetate, dark blobs along the mRNA were seen under the electron microscope. These were interpreted to represent regions of complex secondary structure. Treatment of the mRNA with denaturing reagents prevented the binding of the stain to the message indicating the loss of secondary structure. Favre gt_gl. (64) examined the conformation of duck and rabbit globin mRNA by optical methods and by employing ethidiun bromide to study intercalation of the dye in double stranded regions of the mRNA. These investigators also observed the hyperchromic shift at 260 nm when globin mRNA was heat denatured. In addition, the stimulation of ethidium bromide flourescence following intercalation of the dye into double stranded regions of globin mRNA was measured. The ethidium bromide titration profiles showed that up to 60% of the nucleotides of both duck and rabbit globin mRNA are involved in base pairing interactions, a nunber greater than predicted for a random copolymer conformation. The nucleotide sequences of rabbit a— and B-globin mRNAs were determined during the time that these secondary structure studies were taking place (52,53). The a-globin messenger RNA is 552 nucleotides long and consists of a 50 nucleotide long 5' untranslated region, a 423 nucleotide coding region and a 89 nucleotide long 3' non-coding region. Beta globin mRNA is 590 nucleotides long and is distributed among a 57 nucleotide 5' non-coding region, a 438 nucleotide long coding region and a 95 nucleotide 3' untranslated region. A possible secondary structure map for a-globin mRNA was proposed by Heindell gt al. (53) using the rules for the calculation of interaction energies between bases developed 13 by Tinoco gt a1. (37). This secondary structure is based solely on thermodynamic consideration and may not reflect the actual conformation adopted by the molecule in solution. The previous data on the structure of globin mRNA using physical techniques provided only general information on structural features of globin mRNA. The method of Wurst gt 31. was modified and has been applied to the 5' end of both the a- and B-globin mRNA molecules by Pavlakis gt_gl. (28). The results of secondary structure mapping were combined with the empirical rules of Tinoco to generate structure maps of the 5' ends of both the a-globin and B-globin messenger RNAs, providing the first detailed structure maps for the 5' ends of both messages. The results of their studies showed considerable base pairing in both molecules. The B-globin mRNA initiator codon is not present in a base paired structure and is both exposed and highly susceptible to nuclease attack. The a initiator codon, however, is present in a region of secondary structure and totally inaccessible to nuclease attack by either 51 nuclease or ribonuclease T1. The inaccessibility of the a initiator codon due to its involvement in secondary structure was postulated to be the reason for the slower initiation rate observed for a-globin synthesis. Nascent Globin Peptide Nonuniformity The rate of synthesis of globins in the reticulocyte has been deter- mined by Lodish and Jacobson to be 200 seconds per chain at 25° (10). This value represents the average time required to synthesize a single polypeptide chain without regard to possible heterogenieties in the local translation rate along the mRNA during assembly of the protein. Studies performed by Protzel and Morris showed that the rate of elongation during 14 the synthesis of a- and B-globin is nonuniform (65). This nonuniformity was demonstrated by analysis of the size distribution of nascent peptides isolated from polysomes synthesizing a- and B-globin in rabbit reticulo- cytes. As a ribosome traverses a mRNA during the process of translation, it carries with it one growing nascent peptide chain. The length of this nascent peptide is an exact measure of the location of a ribosome along the coding sequence of the message. If elongation were a process occur- ing at a uniform rate during protien synthesis, all possible nascent peptide sizes would be represented equally in the nascent chains of poly- somes synthesizing that protein. Protzel and Morris showed that analysis of the size distribution of globin nascent peptides by gel filtration under denaturing conditions revealed a nonuniform distribution of sizes (65). The observed accumulations of nascent peptides along the mRNA represent areas of the mRNA where relatively high ribosome density are found on a time-average basis as compared with other areas of the coding sequence. Chaney and Morris investigated the origins of this nonuniform trans- lation rate (66). These workers attempted to find some component of the reticulocyte lysate cell-free protein synthesizing system whose presence in limiting quantities was responsible for the nascent peptide accumula- tions. Their results failed to find any limitation of components in the reticulocyte lysate cell-free system that could affect the nascent pep- tide size distribution. Translation of purified globin mRNA from rabbit reticulocyte was performed in the wheat embryo derived cell-free protein synthesizing system. Nascent peptides purified from the globin mRNA directed wheat embryo cell-free system displayed a similar distribution 15 of globin nascent peptides to that observed in the reticulocyte lysate (66). This led Chaney and Morris to conclude that the origin of the nascent peptide accumulations lies in the mRNA itself. Chaney and Morris also studied the distribution of nascent peptides on polysomes synthesizing M52 coat protein in rifampicin treated M82 infected g; 9911 (67). Analysis of these nascent peptides by gel filtra- tion under denaturing conditions yielded a nonuniform nascent peptide distribution, but a different distribution than that observed for globin nascent peptides. A correlation was made between the regions of the M52 coat protein genome where ribosomes were accumulating and the locations of secondary structure in that same genome proposed by Fiers gt_al. (22, 23). This led Chaney and Morris to propose that regions of secondary structure in mRNA modulate the rate of ribosome translocation along the mRNA, giving rise to the nonuniform nascent peptide population. Vary and Morris continued the studies into the origin of nascent peptide nonuniformity. This was accomplished by subjecting the fractions isolated from the gel chromatographic analysis of total globin nascent peptides to digestion with trypsin (68). The proportion of radioactivity in the a and a tryptic peptides was used to construct the individual nascent peptide size distributions. The a- and s-globin nascent peptide distributions were found to be different from one another. These investigators also made a correlation between the single stranded regions of $1 nuclease susceptibility in the a-globin message performed by Pavlakis gt 21- (28) and the distribution of s-globin nascent peptides. The regions of S1 susceptibility in the B-globin mRNA correlated with positions of minima in the a-globin nascent peptide distribution. This was in keeping with the original proposal of Chaney and Morris that 16 accumulation of nascent peptides represent areas of secondary structure in mRNA, while minima in the profile could now be correlated with single stranded regions of the mRNA. MATERIALS Cycloheximide, ethyleneglycol-bis-(B-aminoethylether)N,N'~tetra- acetic acid (EGTA), guanidinium chloride, (practical grade), bovine hemin, 2,4-dinitrophenyl alanine, dithiothreitol (DTT), creatine phosphokinase (EC 2.7.3.2), phosphocreatine (diTris salt), diethyl- pyrocarbonate, phenylhydrazine-HCl, Triton X-114 and TRIZMA base were purchased from Sigma Chemical Company, St. Louis, Missouri. Scintilla- tion grade napthalene was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin. Bio-Rad Company, of Richmond, California was the source of Bio-Gel A-0.5M, Bio-Gel P-10, acrylamide, N,N'-methylene-bis- acrylamide, sodium dodecyl sulfate (SDS) and N,N,N',N' tetramethylene- diamine (TEMED). Nembutal (sodium pentobarbital) was obtained from Abbott Laboratories, North Chicago, Illinios. Phosphocreatine (dipotassium salt) was purchased from Calbiochem Company, La Jolla, California. Research Products International was the source of 2,5-diphenyloxazole (PPO) and 1,4 bis-2-(4-methyl-5-phenyloxazoyl)- benzene. Adenosine triphosphate, guanosine triphosphate and oligo (dT) cellulose (Type 7) were purchased from P-L Biochemicals, Milwaukee, Nisconsin. Blue Dextran 2000 and Sephadex 6 series gel filtration media were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Whatman Biochemicals Ltd., Maidstone, Kent, Great Britian was the source of microgranular preswollen diethylaminoethyl cellulose (DE52), carboxymethyl cellulose (CM32) and GF/C glass fiber filters. Sodium 17 18 heparin was purchased from Fisher Scientific Company, Fair Lawn, New Jersey. Micrococcal nuclease (EC 3.1.31.1) and calf intestine alkaline phosphatase (EC 3.1.31.1) were obtained from Boeringer-Mannheim Biochemicals, Indianapolis, Indiana. New England Biolabs, Beverly, Massachusetts was the source of T4 RNA Ligase (EC 6.5.1.3). Nuclease S1 (EC 3.1.4.-) was obtained from Miles Laboratories, Elkhart, Indiana. Pactamycin was a gift of the Upjohn Company, Kalamazoo, Michigan. Sparsomycin was generously donated by Drug and Research Development, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. General Mills, Inc., Minneapolis, Minnesota provided the gift of untoasted wheat germ. Tobacco acid pyrophosphatase was a gift from Dr. C. Vary, Syracuse University, Syracuse, New York. Radiolabeled compounds were purchased from Amersham/Searle Company, Arlington Heights, Illinois (L-[3HJ-Leucine, 60 Ci/mmol, L-[14C]leucine, 300 mCi/mmole, L-[3H]isoleucine, 15 Ci/mmole, Adenosine 5'-[ 32P] triphosphate, 2000 Ci/nmole, and cytidine 3' ,5'[5'-32P]-bisphosphate, 2000-3000 Ci/mmole) and New England Nuclear, Boston, Massachusetts (L-[3HJ-tryptophan, 125 Ci/mmole, and L-[14CJ-tryptophan, 58 Ci/mmole). T4 polynucleotide kinase (EC 2.7.1.78) and NEF 963 aqueous counting cocktail were also obtained from New England Nuclear. Ultra pure sucrose and urea were obtained from Bethesda Research Labs., Bethesda, Maryland. X-Omat (XAR-5) films were from Eastman Kodak Inc., Rochester, New York as was “Stains-All". All other chemicals were of reagent grade or higher quality. METHODS Preparation of Rabbit Reticulocytes New Zealand white male rabbits weighing 6-8 lbs. were made anemic with four daily injections of 2.5% phenylhydrazine-HCl (pH 7.3) prepared in NKM salts (69). On the seventh day after the start of phenylhydrazine treatment, rabbits were injected intravenously via the marginal ear vein with 100 mg of sodium nembutal and 2000 units of sodium heparin. Blood was removed by cardiac puncture using a 100 ml syringe fitted with an 18 gauge needle. The blood obtained was quickly cooled to 0°. The hematocrits of these preparations ranged from 10-17%. All subsequent steps were performed at 0°. The blood was filtered through a double layer of glass wool to remove tissue pieces and the filtrate centrifuged at 4000 x g for 10 minutes. Plasma was removed by aspiration with care taken to remove the buffy coat from the surface of the red cell pellets. The cells were resuspended in a volume of Ringers saline approximately equal to the volume of plasma removed. Cells were then reisolated by centrifugation at 4000 x g x 10 minutes followed by removal of the supernatant layer. The cells were washed by this procedure two additional times. Preparation of the Reticulocyte Lysate Washed rabbit reticulocytes were lysed by swirling the cells with an equal volume of ice cold sterile water for ten minutes. The lysate was centrifuged at 25,000 x g x 20 minutes and the supernatant layer gently 19 20 decanted. Aliquots of the lysate were immediately frozen by injection into sterile glass vials maintained at -40° on dry ice. The lysate aliquots were capped and stored in liquid nitrogen until use. The conditions for protein synthesis in the reticulocyte lysate are shown in Table I. Table 1. Final concentration of the components of the reticulocyte lysate cell-free system. These components were added to 800 pl of lysate yielding a final volume of 950 pl. MgClz 1.4 - 1.6 mM KDAC 75 - 80 mM Hemin 3.2 mM ATP 1.0 mM GTP 0.2 mM Creatine phosphate (dipotassium) 11 mM Amino acids 0.1 times the final concen- tration of Hunt (70) Creatine phosphokinase 100 ug/ml The potassium and magnesium concentrations chosen for use in each lysate were determined on the basis of the levels required to obtain the optimal rate of incorporation of [3H]-leucine into trichloroacetic acid insoluble material. Aliquots of the reticulocyte lysate system were incubated at various concentrations of the ion to be optimized (K+ or MgZ+) in a reaction mixture supplemented with 50 uCi/ml L-[3H]- leucine at 37°. At 10 minute intervals 5 ul aliquots of the incubations were removed and added to 1.0 ml of ice cold water. One ml of 1.0 N NaDH, 10 mM L-leucine was added to each sample. The samples were 21 incubated at 37° for 20 minutes. Four ml of 15% TCA, 10 mM L-leucine, 1.0% H202 were added and the samples incubated at 0° for 45 minutes. The precipitated protein was collected on GF/C filters by suction, washed with 25 ml of 10% TCA, 10 mM L-leucine and dried in scintillation vials at 100° for 45 minutes. After the vials had cooled, 5 ml of a Triton- xylene based liquid scintillation cocktail were added and incorporation of [3H]-leucine into TCA precipitable material was determined. Preparation of the Wheat Germ Cell-Free Protein Synthesizinngystem A wheat germ cell-free protein synthesis system was prepared by a modification of the method of Gallis gt 31. (71). Untoasted wheat germ was obtained from General Mills, Inc., Minneapolis, Minnesota and contained approximately 70% wheat germ and 30% chaff. The wheat germ was ground very gently in order to break it into smaller pieces. The pieces which passed through a #14 Tyler sieve but retained by a #28 Tyler sieve were saved. The contaminating chaff was removed by floating 20 g of sieved wheat germ in 400 ml of chloroform:cyclohexane (15:1) in a 500 ml graduated cylinder. After mild swirling, the chaff settled to the bottom and the embryos were poured off into a coarse glass sintered funnel. The solvent was removed by aspirator suction, after which embryos were air dried overnight and stored at -20°. Preparation of a wheat embryo S-23 fraction involved suspending 5 grams of embryos in 8 ml of grinding buffer (1 mM Mg(0Ac)2, 2 mM CaClz, 50 mM KCl and 1 mM dithiothreitol) with an equal weight of acid washed glass beads in a chilled mortar. The mixture was ground hard in the cold for 2 minutes. The embryos were ground with 8 ml additional grinding buffer for 2 more minutes in the cold, and the pasty mixture transferred to a sterile centrifuge tube. The material adhering to the 22 mortar was rinsed into the centrifuge tube with 9 ml of grinding buffer and the material in the tube thoroughly mixed with a sterile scoop spatu- la. The cell lysate was centrifuged for 10 minutes at 23,000 x g and the supernatant fluid decanted into a prechilled sterile centrifuge tube. The volume of the extract was estimated by adjacent tube volume, 0.01 volume of 100x buffer (1 M Tris-HCl pH 7.5, 100 mM Mg(0Ac)2) added and the extract centrifuged for 10 minutes at 23,000 x g. The supernatant layer was removed and passed over a 2.5 x 40 cm Sephadex G-25 (fine) column equilibrated with 20 mM HEPES pH 7.5, 40 mM KCl, 2.0 mM Mg(0Ac)2 and 1 mM DTT. The void volume fractions with the highest absorbance at 260 nm were pooled and the absorbance of this pooled S-23 fraction was measured at 260 nm. If the A250 were greater than 75 Azso/m" the S-23 fraction was diluted with column buffer to an absorbance of 75 A250/ml and frozen in a dry ice-acetone bath in 400 pl aliquots, or frozen directly if the A250 were less than 75/ml. Determination of the Rate of mRNA Directed Protein Synthesis in the Wheat Germ Cell-Free System Incubations for the determination of protein synthesis in the wheat germ system were performed in a volume of 20 ul or multiples thereof. The conditions employed for protein synthesis in the wheat germ system are shown in Table II. Each 50 ul reaction mixture contained 30 ul of the S-23 fraction. Incubations were performed at 28°C. At specified time intervals, 2 ml of 0.5 N NaDH, 0.25 mg/ml BSA and 10 mM L-leucine were added to each tube in order to terminate protein synthesis. Radio- activity incorporated into TCA precipitable material was determined as described above for reticulocyte lysate incubations but with H202 omitted in the 15% TCA used for protein precipitation. 23 Table II. Final concentrations of components added to the wheat germ cell-free system. HEPES* 12 mM Mg(0Ac)2 3.0 mM KDAC 140 mM ATP 1.6 mM GTP 0.16 mM Phosphocreatine (diTris) 12.8 mM Amino acids 32 uM mRNA 0-5 ug/ml *present in S—23 preparation Preparation of the mRNA Dependent Reticulocyteggysate Cell-Free Protein Synthesizing System A mRNA dependent reticulocyte lysate was prepared by a modification of the procedure of Pelham and Jackson (72). A complete lysate cell-free protein synthesizing system was assembled as described earlier. The lysate was made 1 mM in CaClz and 20 ug/ml in micrococcal nuclease, mixed thorougly and incubated at 20°C for 15 minutes. Sufficient 400 mM EGTA was then added to give a final EGTA concentration of 2 mM. After mixing, the mRNA dependent lysate was frozen in glass vials on dry ice in 400 pl aliquots and stored in liquid nitrogen. In some cases, the amino acid component was lyophilized before the lysate system was assembled in order to allow larger volumes of mRNA to be added without changing the final concentrations of the components of the system. In order to determine the ability of the mRNA dependent lysate to synthesize protein in response to exogenous mRNA, aliquots of these lysates were thawed at 24 4°. The lysate was either supplemented with increasing amounts of mRNA up to a final concentration of 5 ug/ml or H20 if a measurement of the background synthetic rate was to be made. The mRNA dependent lysates were incubated at 37° and assayed in the same manner as described above for the reticulocyte lysate. Preparation of Rabbit Globin mRNA Rabbit globin mRNA was prepared by a modification of the method of Krystosek gt_al, (73). Washed rabbit reticulocytes were prepared as described above, but with 1 mM cycloheximide added to the Ringers saline. The cells were lysed by the addition of four volumes of RSB (10 mM KCl, 1.5 mM MgCl2 and 10 mM Tris-HCl pH 7.5) followed by incubation with gentle swirling at 0° for 5 minutes. The lysate was centrifuged at 25,000 x g for 15 minutes. The supernatant fraction was carefully trans- ferred by pipet into ultracentrifuge tubes and centrifuged for 2.5 hours at 252,000 x g in a Beckman 60 Ti rotor. The supernatant fluid was dis- carded and the ribosome pellets used to prepare globin mRNA either immed- iately or the pellets were frozen at -80° for preparation of mRNA at a later time. The pellets were resuspended in HSB (High Salt Buffer, 1 mM EDTA, 150 mM NaCl and 10 mM Tris-HCl pH 7.5) and the suspension centri- fuged at 16,000 x g x 10 minutes. The supernatant fraction was adjusted to 50 A250/ml with HSB. The solution was warmed to room tempera- ture and made 0.5% in SDS. After 5 minutes of standing, the RNA solution was added to a 30 ml Corex tube containing 1 gram of oligo (dT) cellulose which had been equilibrated with HSB. This slurry was mixed gently on a rocking shaker for one hour to allow the poly(A) containing fraction to hybridize to the oligo (dT). The slurry was poured into a sterile 1.0 x 10 cm glass column. The oligo (dT) cellulose column was washed with HSB 25 containing 0.5% 505 until A260 of the eluate fell to less than 0.05. The column was then washed with 2 volumes of HSB to remove SDS and the poly(A) containing RNA was then eluted from the column with H20. The fractions containing the RNA were pooled, made 1 mM in EDTA and heated for 10 minutes at 65°C. The solution was made 150 mM in NaCl and re-ap- plied slowly to the oligo (dT) cellulose column. The column was then washed with HSB until the A250 of the eluate fell to 0.02. The mRNA was eluted with H20, pooled, and made 2% in‘KOAc. Two volumes of ethanol were added and the mixture allowed to stand at -20°C overnight to precipitate the messenger RNA. The precipitated mRNA was collected by centrifugation at 9000 x g x 20 minutes at 0°. The pellet was dissolved in a minimal volume of H20 and the absorbance of the solution at 260 nm determined. The RNA solution was adjusted to a concentration of 1 mg/ml (assuming 20 A250 units/ml = 1 mg/ml RNA) and stored at -80° in small aliquots. Identification of Products from Cell-Free Protein Biosynthesis: Separation of a- and a-Globin The identification of the products synthesized by the reticulocyte lysate cell-free system or by either of the mRNA dependent cell-free systems was done by ion exchange chromatography using CM cellulose according to a modification of the procedure of Dintzis (74). Aliquots of postribosomal supernatant fractions of [3HJ-labeled lysates which had been centrifuged at 105,000 x g x 150 minutes to remove ribosomes were added slowly to 30 volumes of 0.6 N HCl in acetone maintained at -40° in a dry ice-acetone bath. Following 45 minutes of stirring, the globin was recovered by centrifugation at 13,000 x g x 30 minutes. The pellet was dissolved in Dintzis buffer (0.02 M pyridine, 0.2 M formic 26 acid, 0.05 M 2-mercaptoethanol) and dialyzed overnight against two 1 liter portions of the same buffer. The dialyzed solution was centrifuged at 30,000 x g x 20 minutes to remove insoluble material. A solution containing approximately 100,000 cpm was loaded onto a 1 x 25 cm CM32 cellulose column previously equilibrated with Dintzis buffer. The column was washed with 10 column volumes of Dintzis buffer and then eluted with a pyridinium formate gradient as previously described (75). Three ml eluate fractions were collected directly into scintillation vials and counted after addition of 10 ml of Formula 963. Polysome Labeling for Preparation of Peptidyl-tRNA Labeling of peptidyl-tRNA was performed in 950 pl incubations in the case of reticulocyte lysates, 400 u] for mRNA dependent reticulocyte lysates and 500 pl for wheat germ lysates. Incubations for wheat germ lysates were performed at 28° for 20 minutes while the reticulocyte lysates were incubated for 10 minutes at 37°. The labeling reactions were terminated by addition of two volumes of Medium B (75) containing 210 uM Sparsomycin and 59 uM cycloheximide (medium 8 and antibiotics). Polyribosomes were isolated by centrifugation at 105,000 x g x 150 minutes at 2-4°. Peptidyl-tRNA Purification The polysomal pellet was resuspended in 0.75 ml of 0.25 M sucrose, 210 uM Sparsomycin, 59 pH cycloheximide. The suspension was made 3.0 M in LiCl, 4.0 M in urea, 50 mM in 2-mercaptoethanol and 50 mM in NaDAc, pH 5.6 in a final volume of 2.0 ml. The mixture was incubated at 0° for 16 hours after which time it was centrifuged at 25,000 x g x 20 minutes. The supernatant fraction was applied to a 1.5 x 45 cm Bio-Gel P-10 column equilibrated with Buffer I. The column was eluted with Buffer I and the 27 radioactive material eluting in the void volume applied to a 1.0 x 2 cm DE52 cellulose column equilibrated with Buffer I. The DE52 column was washed with 100 volumes of Buffer I following which peptidyl-tRNA was recovered by stepwise elution of the colunn with Buffer II. The fractions eluted with Buffer II containing radioactivity were pooled and concentrated by ultrafiltration in an Amicon BMC ultrafiltration cell (Amicon Corp., Lexington, Massachusetts) using an Amicon UM-2 membrane. When the sample had been concentrated to a volune of 0.3—0.5 ml, 3 ml of 6 M guanidinum chloride, 100 mM 2-mercaptoethanol pH 6.5 were added and the solution again concentrated to 0.5 ml. The concentrated peptidyl- tRNA was made 0.3 M in Na0H and incubated at 37° for 4 hours in order to cleave aminoacyl and peptidyl-tRNA bonds. The solution was then adjusted to approximately pH 5.6 with 6 N HCl as determined by pH paper and stored at -20° until analyzed. Preparation of the Bio-Gel A 0.5m Column and Analysis of Nascent Peptide Size Distribution The size distribution of nascent peptides was analyzed by gel filtration chromatography as described by Protzel and Morris (65). A slurry of 225 ml of Bio-Gel A 0.5m was brought to a final volume of 1 liter with deionized water in a graduated cylinder. The gel was allowed to settle to 40% of the original column height followed by removal of the upper layer by aspiration. This sequence was repeated twice. The slurry was then allowed to settle completely and the supernatant fluid was removed. The slurry was decanted into a 1 liter flask and sufficient solid guanidinum chloride slowly dissolved in the slurry to make 500 ml of 6 M guanidinum chloride solution. The slurry was then made 100 mM in 2-mercaptoethanol, titrated to pH 6.5 with 1 N HCl and brought to a 28 volume of 500 ml in a graduated cylinder. The slurry was de-gassed and poured in 1.5 x 100 cm Pharmacia analytical column to which sufficient 6 M guanidinum chloride had been added to fill the lower 20 cm of the column. Following 10 minutes of standing, the column was allowed to pack under a constant hydrostatic head of 50 cm. When the bed height had reached 90 cm, packing was stopped and the column washed with 2 column volumes of guanidinum chloride. The column was run for a minimum of 24 hours before application of a sample. A sample to be analyzed was made 50 mM in dithiothreitol and 8% in sucrose. After two hours at room temperature, 60 pl of 3.6% Blue Dextran in 6 M guanidinum chloride and 40 pl of 0.2% DNP-alanine in 6 M guanidi- num chloride were added to act as markers for the excluded and included volumes. The sample was applied to the column through a column of running buffer by layering the sample onto the top of the gel bed with a pasteur pipet. The sample was analyzed by gel filtration chromatography at a flow rate of 6 ml/hr and 0.9 ml fractions collected directly into scintillation vials for determination of radioactivity. Nascent peptide distributions were constructed by counting of 120 fractions which eluted between Kd=0 and Kd=1. Cyanogen Bromide Cleavage of a- and s-Globin Cyanogen bromide cleavage of globin was performed in a fume hood. [14CJ-labeled rabbit globin was dissolved in 70% formic acid at a concentration of 5 mg/ml. Cyanogen bromide was added to a 400 fold molar excess with regard to the methionine present. The solution was incubated in the dark for 48 hours at room temperature. After 48 hours of reaction the mixture was diluted with 10 volumes of water and lyophilized. 29 Determination of Distribution Coefficients Elution data obtained from the elution of products during Bio-Gel A 0.5 m gel filtration chromatography in 6.0 M guanidium chloride were treated as described by Fish and Tanford (76). The distribution coefficient, Kd, was calculated from the formula: Kd = (Ve ' Vo)/(Vl ' Vo) where Ve = mass of solvent corresponding to the peak concentration of eluting solvent. V0 = column void volume in mass of solvent. V] = mass of solvent contained within the column and the gel matrix. In this work, parameters were determined using the volume rather than the mass of the solvent. Blue Dextran 2000 was used as a marker for the void volune and DNP- alanine employed as a marker for the volume of colunnlwhich was accessible to solvent. The eluate fractions containing Blue Dextran and DNP-alanine were identified by visual inspection of the fractions. Kd values were then assigned to intermediate fractions by their relative positions between these markers for Kd 0 and Kd 1.0, respectively. Recrystallization of Guanidinium Chloride Guanidinium chloride was recrystallized by a modification of the method of Nozaki and Tanford (77). One kilogram of practical grade guanidinium chloride was dissolved in sufficient absolute ethanol at 75° with stirring to yield 3.5 l of solution. One gram of activated charcoal was added to the solution followed 5 minutes later by 1 gram of celite. The solution was then filtered hot through Whatman No. 1 filter paper in 30 an 18.5 cm Buchner funnel using a gentle vacuun. The filtrate was heated to redissolve any crystals which may have formed during filtering and sufficient toluene was added to induce crystal formation. The solution was allowed to cool to room temperature and then placed at 4° overnight. The crystals were harvested by filtration in an 18.5 cm Buchner funnel, washed with -20° ethanol and air dried. The dried crystals were dissolved in a minimum volume of absolute methanol at 70°. When complete dissolution was achieved, the solution was allowed to cool to room temp- erature and then placed at -20° overnight. The crystals which formed were harvested as before and dried to constant weight ig_ygggg. Preparation of Stock Urea, Buffer I, Buffer 11,,and DE52 Stock urea was prepared by deionizing a solution of 9.0 M urea by stirring with Amberlite MB-3 for at least 4 hours. The Amberlite was removed by filtration through a medium grade sintered glass funnel and the volume of the resulting filtrate adjusted to yield a solution with a final urea concentration of 8.54 M. This stock urea was used to make Buffers I and II for peptidyl t-RNA purification as described by Chaney and Morris (66). DEAE cellulose (DE52) was suspended in 0.5 N HOAc (10.5 g DE52/100 ml 0.5 N HOAc). C02 was removed by agitating the suspension under reduced pressure. The pH of the slurry was adjusted to 5.6 with saturated Na0H and the suspension was allowed to settle to one half its original column height. "Fines" were removed by aspirating off the supernatant layer. The cellulose was resuspended in Buffer II and fines removed as before followed by two more washings in Buffer I. 31 Separation of Rabbit a- and geGlobin mRNAs Rabbit globin mRNA was separated into its a and 3 components by preparative polyacrylamide gel electrophoresis using a Bio Rad agarose gel bridge. The gel was composed of 2.6% acrylamide wih a bisacrylamide to acrylamide ratio of 1:20 in 7 M urea. To prepare a gel for the separation of mRNA the legs of the gel bridge were filled with a 4.5% acrylamide gel in 7 M urea. The gel legs were prepared by mixing 22.5 ml of 40% acrylamide (acrylamidezbis of 20:1), 20 ml of 10 x TBE ( 1 X TBE = 90 mM Tris, 80 mM Boric acid and 2.5 mM NazEDTA) and 155.5 ml 9 M deionized urea. The solution was de-gassed, made 0.08% in ammonium persulfate, and 0.08% in TEMED and poured into the legs of the gel. The horizontal slab gel for the separation of mRNA was prepared by mixing 243 ml 9 M urea, 25 ml 31.5% acrylamide and 30 ml 10X TBE. Following removal of dissolved gasses, the acrylamide solution was supplemented with ammonium persulfate and TEMED to 0.08% and poured into the gel bridge. The horizontal polyacrylamide slab gel measured 15 x 21 x 1 cm. After the gel had polymerized for one hour it was placed at 4° to cool until use. The RNA sample was made 7 M in urea by addition of RNAase free ultra pure urea. The sample was heated to 65° for 5 minutes and quick cooled; 0.1 vol we of 10X TBE and 0.05 vol me of 0.1% bromophenol blue in 50% glycerol were then added. The sample was loaded into the gel as a single band in a 1 mM x 1 cm x 10 cm sample slot made in the gel during casting. Electrophoresis was performed using a Bio-Rad model 1405 horizontal electrophoresis system, maintained at 20°, at a constant 100 V. Electrophoresis was stopped when the dye front had migrated the full 15 cm to the edge of the gel. The gel was removed from the bridge and stained with 2 pg/ml ethidium bromide in 0.2 M KCl until 32 the a- and B-globin mRNA bands could be seen under long wave UV light using a UVSL-25 mineralite lamp (Ultraviolet Products, Inc., San Gabriel, California). The bands were excised with a razor blade. The RNA was recovered by electrophoretic elution using a modification of the procedure of Weinand gt 11. (78). A horizontal 1% agarose gel in TBE was prepared in a 12 x 20 x 1.2 cm plexiglass tray. Two rectangular shaped holes of twice the width of the gel slice to be eluted were cut out of the gel. These slots were lined with sterile dialysis tubes which had been slit open at one side and knotted at both ends making sure the dialysis bags contacted all sides of the holes. A gel slice was placed in each bag, positioning the gel slice closest to the cathode. The remainder of the hole was filled with TBE to the upper edge of the gel slice. The agarose gel was connected by 3 MM paper wicks to the buffer chambers of the Model 1405 electrOphoresis unit and electrophoresis performed overnight at 200 V. After electrophoresis the buffer in each of the gel slots was thoroughly mixed making sure that the portion of the dialysis bag nearest the anode was well agitated. The buffer was then removed, placed in a sterile Corex centrifuge tube and extracted with n-butanol to insure removal of all ethidium bromide. The buffer was then made 150 mM in NaCl and the mRNA bound to a 0.50 ml oligo (dT) cellulose column in a pasteur pipet. The column was washed and the RNA eluted as described above. Translational Purity of Rabbit a- and B-Globin mRNAs The translational purity of the a- and B-globin mRNAs was determined by the ratio of a- and e-globin synthesized when each mRNA was translated in either the nuclease inactivated lysate or the wheat germ lysate. An aliquot of the appropriate lysate was made 5 pg/ml in either a- or 33 B-globin mRNA and 100 pc1/mi in a radiolabeled amino acid. The incubation was allowed to proceed for 30 minutes followed by centrifugation of the incubation mixture for 2 hours at 105,000 x g at 4°. The supernatant fractions were analyzed for the amount of a and a globin synthesized by chromatography on CM cellulose as described above. Limited Nuclease S1 Digestion of Globin mRNA Ly0philized nuclease 51 was dissolved in $1 Buffer (50 mM Na0Ac pH 4.5, 200 mM KCl and 1 mM ZnClz) to a final concentration of 106 units/ml and stored at 4°. This solution was used as a working stock. Globin mRNA was made 1X in S1 Buffer by addition of one tenth volume 10X 51 buffer and the solution adjusted to an RNA concentration of 250 pg/ml. Nuclease S1 was added to a final concentration of 250 units/ml and the reaction mixture incubated at 37°. At 5 minute intervals, aliquots of the mixture were withdrawn and the reaction terminated by addition of 0.1 volume of 0.5 M Tris-HCl pH 7.5, 0.02 M EDTA. Samples were stored at -80° until used. 3' End Labeling of Globin mRNA The reaction conditions for the 3' labeling of globin mRNA are a modification of those used by 3' end labeling of RNA by England gt El- (79). The components of the 3'labeling mixture are shown in Table III. 34 Table III. Final concentration of Components in the 3' Terminal Labeling of Globin mRNA. HEPES pH 8.3 50 mM MgCl2 10 mM Dithiothreitol 3.3 mM ATP 6 pmM Bovine Serum Albumin 10 pg/ml Dimethyl Sulfoxide 10% (v/v) 5'[32P]-pCp (2000-3000 Ci/mmole) 1.0 uM mRNA 200 pg/ml The concentration of mRNA in the reaction was approximately 1 pM. The reaction was allowed to proceed at 4° for 16 hours. The reaction was terminated by the addition of SDS and NaCl to final concentrations of 0.5% and 150 mM respectively. Unreacted pCp and any other low molecular weight impurities were removed by application of the reaction mixture to a small oligo (dT) cellulose column made in a pasteur pipet. The column was washed with HSB until no 32P was detected in the column effluent. Labeled globin mRNA was then eluted from the column with H20, made 2% in KOAc pH 5.5 and precipitated with 2 volumes of ethanol. The mRNA was collected by centrifugation for 15 minutes in an Eppendorf 5412 table top centrifuge. The pelleted RNA was dissolved in water to a final concentration of 250 pg/ml as determined spectrophotometrically at 260 nm. 5' End Labeling of Globin mRNA The 5' end of globin mRNA was labeled in a three step reaction based on a modification of a previous scheme (80). The 7-methyl guanosine of 35 the cap structure was removed with tobacco acid pyrophosphatase (TAP), the 5' end dephosphorylated with calf alkaline phosphatase, and the 1-[32PJphosphate transferred from ATP to the 5' terminus of globin mRNA using T4 polynucleotide kinase. For removal of the m7G, 10 pg of globin mRNA were ethanol precipitated and redissolved in 20 pl of 50 mM Na0Ac pH 6.0 freshly made 10 mM in 2-mercaptoethanol. One unit TAP was added and the mixture incubated at 37° for 30 minutes. Three pl of 0.5 M Tris-HCl pH 8.3 (at 37°), 1 pl of 5 units/ml calf alkaline phosphatase and sufficient water to bring the volune to 30 pl were added to the mixture. Dephosphorylation was carried out for 15 minutes at 37°. Two pl of 200 mM potassium phosphate pH 9.5 were added, followed by thorough stirring. The mixture was transferred to a tube containing dry v>[3ZP]-ATP. To this were added 2 pi of 0.2 M MgClz, 4 pi of 40 mM dithiothreitol, and 2 pl of 2000 units/ml T4 polynucleotide kinase. The mixture was incubated for 30 minutes at 37°. The labeled mRNA was precipitated by addition of 100 pl 2 M NH40Ac and 500 pl of ethanol; a procedure which precipitates RNA without precipitation of the proteins present in the mixture (33). The 5' end labeled mRNA was redissolved in HSB and repurified by chromatography on a Sephadex G-25 colunn to remove unincorporated label. RESULTS Wheat Germ Cell-Free System In order to study the nonuniform accumulation of nascent globin peptides, globin mRNA purified from rabbit reticulocytes was translated in the wheat germ cell-free protein synthesizing system. In this manner, a determination could be made of whether or not the observed accumulations of nascent chain populations of discreet size classes were a function of the reticulocyte lysate biosynthetic system or some unique function associated with the globin messenger RNA itself. The wheat germ cell-free protein synthesizing system was chosen for the translation of purified globin mRNA for three reasons. First, this system was known to contain an extremely low level of endogenous messenger RNA activity and would respond well to exogenous mRNA. Second, the products synthesized in the wheat germ cell-free system could be labeled to a high specific radioactivity due to the ability to remove endogenous amino acids. Third, the wheat germ system was sufficiently phylogenetically distant from the rabbit reticulocyte system to provide a good comparative system for the study of the origion of the nascent peptide accumulations. The wheat germ cell-free system was prepared by the method of Gallis .E£.21- (71). Figure 1 shows the incorporation of [3H]-leucine into TCA precipitable material in response to added globin mRNA. These data show that the system responded to added mRNA by incorporating the labeled amino acid into TCA precipitable material in a linear fashion for one 36 37 FIGURE 1. The incorporation of [3HJ-leucine into trichloracetic acid precipitable material by the wheat germ cell-free protein synthesizing systan in the absence of added mRNA (CD) or with 5 pg/ml added rabbit globin mRNA (0). l0 cpm x 3H 38 T T I r ‘ 45 Ci ‘0 ‘1’: I 1 1 IS 30 45 60 Time (min) 39 hour. The rate of protein synthesis displayed an optimum at 3.0 mM Mg(0Ac)2 (Figure 2A) and 140 mM KOAC (Figure 2B). The rate of incor- poration of labeled amino acid was dependent on the quantity of added mRNA until the system became saturated at a mRNA concentration of 10 pg/ml (Figure 3). All wheat germ incubations were performed at a concen- tration of 5 pg/ml of added mRNA in order to insure that the mRNA concentration was limiting. Purification of Rabbit Globin mRNA The mRNA used in these preliminary experiments was prepared by the method of Aviv and Leder (81). The method was found to yield low amounts of globin mRNA which were still highly contaminated with both 18S and 285 ribosomal RNA. Therefore, globin mRNA was purified by a modification of the "quick“ method of Krystosek gt al. as described in Methods. Figure 4 compares the rate of incorporation of globin mRNA purified by the two different methods. The mRNA prepared by the quick method of Krystosek directed incorporation of amino acids into polypeptides at twice the rate observed with mRNA purified by the method of Aviv and Leder. The doubl- ing of the rate of synthesis was due to removal of rRNA found to contam- inate mRNA prepared by the method of Aviv and Leder. All experiments were performed with globin mRNA purified by the method of Krystosek. Characterization of the Product Synthesized in Response to Globin mRNA A wheat germ lysate containing [3H]-tryptophan was allowed to synthesize protein in response to added globin mRNA for 1 hour. Authent- ic [14CJ-globins were added to the incubation and polysomes were removed by centrifugation. Globins were prepared by the acid-acetone method and analyzed by CM cellulose chromatography as described in Methods. The elution profile shown in Figure 5 demonstrates that the 40 .Amm meamwmv :oFHmLucmucou accumum sawmmmuoa mg» can A_uc< .u mmawmu 0.0 #0 N0 0.0 _ mum He E-O' x wdp ) 53 observed, it is the position of the peak within the profile rather than its absolute magnitude which maps ribosome position on the message. Calibration of the Bio-Gel A 0.5m Column Fish and Tanford described a linear relationship between the Kd1/3 and (molecular weight)-555 for the elution of proteins from agarose based gel filtration columns using 6 M guanidinium chloride, 0.1 M 2-mercaptoethanol, pH 6.5 as a solvent (76). This relationship was verified for the elution of proteins from Bio-Gel A 0.5m by Protzel and Morris. The [14CJ-labeled cyanogen bromide generated fragments of rabbit globins (aCNBRl, BCNBRl) were used to calibrate the Bio-Gel A 0.5m column. Cleavage of [14CJ-tryptophan labeled rabbit a- and 3- globin with cyanogen bromide generates only 2 labeled fragments. Calibration of the Bio-Gel A 0.5m column was accomplished using a mixture of [14C] B-globin, aCNBRI and BCNBRZ supplemented with Blue Dextran and DNP-alanine as molecular weight markers. Figure 8 shows the elution of these molecular weight markers from the column. Upon analysis of the data, the relationship of Kd1/3 vs. MW -555 was obeyed by the molecular weight markers. The data for the elution of the markers were fitted to a line by linear least squares regression and yielded the equation: Kd1/3 = (-2 x 10'3)MW-555 + 1.03 This relationship was used to calculate the average molecular weight of the peptides represented by the peaks and valleys in the nascent peptide distribution. Methylmercuric Hydroxide Treatment of Globin mRNA Methylmercuric hydroxide has been shown to be an effective denaturant for both DNA and RNA (82). Concentrations of 2.5 mM or 54 pame_ pee 2: .czozm mumu as» Low mmm.Azzv .m> m\Hc¥ we “e_a mg» mzozm .mzoeem nuwz coxems mew «crampmuazo use :mcuxmo mapm yo mosspo> cowu:_m .:_no_m-m use .5 mo mucmEmmem mu_soea cmmocmxo um_wnw_ cmzaouaxguuHQVHu saw: cespou cowumgu—ww pom pmuwux—mcm Em.o < Fmonowm wzu mo cowumgnw_mu .w mmawmu 55 nx 0.. 90 md ed «.0 o.o . q _ _ _ > ‘ a e_olmzo 5:380 35 one; .2 EN 2.. on. om _ fi q — 1 we Mn 1 0.0 P4 c. .. o._ _ b L _ _ x UJdO Dbl 0| 56 greater have been shown to denature nucleic acids as evidenced by alteration of electrophoretic mobility and by changes in Optical properties of the polymers. Payvar and Schimke have reported the enhancement of the translation of both ovalbumin and conalbumin mRNA in the mRNA dependent lysate following treatment of the mRNA with 2.5 mM methylmercuric hydroxide (83). Denaturation in this manner also changed the rate of synthesis of cDNA by reverse transcriptase as well as the length of cDNA transcript synthesized. These effects were attributed to secondary structure alteration of the mRNA upon denaturation. Experiments involving the translation of methylmercuric hydroxide treated globin mRNA in either the wheat germ cell-free protein synthesiz- ing system or the mRNA dependent reticulocyte lysate were performed. These experiments were undertaken in order to determine if methylmercuric hydroxide denaturation of the globin mRNA would generate an altered nascent peptide distribution upon its translation. The following procedure was performed in a fune hood. Methylmer- curic hydroxide was added to globin mRNA to a final concentration of 0 to 5 mM. Control mRNA was treated with equivalent volumes of H20. The mRNA samples were incubated at room temperature for 5 minutes after which time the mRNA was immediately added to the appropriate protein synthesiz- ing system. The ability of methylmercuric hydroxide denatured globin mRNA to direct the synthesis of protein in either the mRNA dependent lysate or wheat germ cell-free system supplement with [3HJ-leucine is shown in Figure 9. Treatment of globin mRNA with concentrations of methylmercuric hydroxide up to 2.5 mM has no effect on the incorporation of 57 .25 e.m. I ”2... 2a. 0 use .3. d use 8;. D “£853 2.: e .nunwe mcewueeuceeeee eewxeeex; eweeeeeewxgeee we eem uceemec cweewm esp :e 98% reduction of synthesis was attained. It is interesting to note that the rate of inactivation of the lysate was non-linear and followed a time course similar to that observed for the $1 nuclease digestion of purified globin mRNA. When supplemented with globin messenger RNA, the mRNA dependent reticulocyte lysate incorporated labeled amino acid into TCA precipitable material for greater than 40 minutes at 37°. There was minimal incorporation of label into TCA precipitable material in the absence of added mRNA (Figure 16). The rate of protein synthesis was dependent on the quantity of added messenger RNA until saturation of the system was achieved at 12 pg/ml of mRNA. Nascent Peptide Analysis from the mRNA Directed Synthesis of Globin in the mRNA Dependent Reticuloeyte Lysate A 400 pl aliquot of the mRNA dependent reticulocyte lysate was made 5 pg/ml in globin mRNA and nascent peptides were labeled with [3H]- tryptophan for 10 minutes at 37°. Protein synthesis was halted and the 75 FIGURE 15. The time course of micrococcal nuclease inactivation of endogenous protein biosynthetic activity in the reticulocyte lysate. cpm xl0-4 3H 76 l0 l2 :‘t 1'5 Digesfion Thne(nwn) 20 Tinie 30 4E) 50 (nun) 77 FIGURE 16. The globin mRNA directed incorporation of [3H]-leucine into TCA precipitable material in the mRNA dependent lysate. No added mRNA (0 ), 5 pg/ml globin mRNA( 0 ). cpm x l04 3H 78 5.0- 2.5- __n J J 20 Time (min) 79 incubation mixture combined with an intact reticulocyte lysate whose nascent peptides had been labeled with [14CJ-tryptophan. Nascent peptides were co-purified and chromatographed on a Bio-Gel A 0.5m colunn. Analysis of the combined nascent peptides is shown in Figure 17. The size distribution of nascent peptides shows a surprising difference when compared to that observed in the intact lysate. The shoulder seen in the intact lysate at Kd 0.58 is now a major peak in the profile and the peak at Kd 0.71-0.72 has also become enlarged. The remainder of the distribution shows a similar distribution of components although the high molecular weight region has been reduced in relative amplitude. An analysis of the nature of the products synthesized by the mRNA dependent lysate was performed on the labeled globin present in the post- ribosomal supernatant fraction from this incubation. Authentic [14CJ-globins resulting from the synthesis of globin in the [14CJ-labeled control reticulocyte lysate were already present in the postribosomal supernatant fraction and were used as markers. Globin was prepared from the postribosomal supernatant fraction by acid-acetone precipitation and analyzed by CM cellulose chromatography as described in Methods. The results are shown in Figure 18. The 0:8 ratio of 0.96 calculated from the areas under the a- and B-globin synthesized in the reticulocyte lysate. The «:3 ratio observed for the translation of globin mRNA in the mRNA dependent lysate was calculated to be 0.79. This imbalanced globin synthesis was reproducibly observed in several different mRNA dependent lysate preparations as well as with different preparations of globin mRNA. These same globin mRNA preparations synthesized o- and a-globin in the wheat germ system at a ratio of 0.92-0.95. The ratio of a- to s-globin mRNA in intact reticulocytes is 80 .euemaw maxeeweewuew eeue>wueecw emeewesc weeeeeewewe eeueewwe c0235 00— - x wdo “2 0| 84 approximately 1.4:1 (10). Beta globin mRNA, however, is known to initi- ate protein synthesis at a rate that is 30-40% greater than for the a-globin mRNA (10). The higher rate of 3 mRNA initiation results in an approximate 1:1 ratio of a- and B-globin synthesis observed in intact reticulocytes. The digestion of endogenous globin mRNA with micrococcal nuclease has somehow altered the mechanisms in the reticulocyte by which the a:3 ratio is maintained. Analyeis of Background Nascent Peptides in the mRNA Dependent Reticulgeyte Lysate in the Absence of Added mRNA It can be seen from Figure 16 that the mRNA dependent lysate shows no detectable incorporation of amino acids into TCA precipitable material in the absence of added mRNA. The mRNA dependent lysate was, therefore, analyzed for the presence of a nascent peptide fraction in the absence of added mRNA. These experiments determined if changes observed in the size distribution of nascent chains isolated following the translation of globin mRNA in the mRNA dependent lysate was attributable to the added mRNA or was some function of the system used to translate the mRNA. An aliquot of the mRNA dependent lysate was supplemented with [3HJ-tryptophan but no mRNA, and incubated for 10 minutes at 37°. Protein synthesis was terminated, ribosomes were isolated by centrifugation, and nascent chains were prepared. The result of one such analysis is shown in Figure 19. The presence of two peaks of accumulation at Kd 0.58 and 0.71 are seen in the size distribution of background nascent peptides. These peaks, as well as other minor accumulations, reveal the source of the gross enlargement of peaks within the nascent chain profile obtained when translating globin mRNA in the mRNA dependent lysates. It is interesting 85 .ewemxw epxeeweewuew uceeceeee e xwweweweEEee ecu eeww meewueee eceeme: eeweeew ecweeew we ewwweee :ewpewe egw .NN mmzwww 96 LO 97 .wpeecw mmemweec weeeeeewewe emwemwwe wueweemwe we mewewwm .mcewueeww .vm maswww 126 I l I l l l _ 127 region of overlap between mRNAs in the gel, individual messenger RNAs of high purity were obtained. It was decided that the sacrifice of yield in RNA was justified in order to obtain high purity a- and s-globin mRNA globin mRNA fractions in a single electrophoretic step. Vournakis et 31. (28) have since reported a complex two dimensional electrophoresis system for separation of a- and a-globin mRNA. However, the feasibility of this method for large scale preparation is not known. Assessment of the Translational Purity of o- and B-Globin mRNA Figure 35 shows the electrophoretic separation of a— and s-globin mRNA together with unfractionated total globin mRNA. Each can be seen as a narrow migrating band with no apparent contaminating bands. The translational purity of each message was determined by its translation in the wheat germ cell-free system followed by analysis of the products synthesized by chromatography on CM cellulose to determine the relative amounts of a- and e-globin synthesized. A 0.5 pg aliquot of a-globin mRNA was incubated in a 100 pl wheat germ cell free incubation mixture supplemented with [3HJ-isoleucine for 60 minutes and centrifuged to remove polysomes. Following ultracentrifugation, the supernatant fraction was analyzed for the amount of a- and B-globin that was present. Figure 36 shows the result of one such separation. From the peak areas and correcting the peak areas for the number of isoleucines present in a- and B-globin, it was determined that the a-globin mRNA fraction directed the synthesis of >95% a-globin. Uniformly labeled [14CJ-globins were included to verify the positions of authentic d- and B-globin. The result of an analysis of the products synthesized by the B-globin mRNA fraction in the wheat germ system supplemented with [3H]-tryptophan is shown in Figure 37. For this fraction, 93% of the counts appear in the 128 FIGURE 35. Gel electrophoresis of globin mRNA: A, total globin mRNA: B, a-globin mRNA: C, B globin mRNA. 129 130 . Al lvmudm mcweewmum use .5 cezeeuexwu- mmueewecw zewwe mgw .Emumam ELm w .A.||.vm:mu .meweecepm wecemucw we emeewecw mew Hg .ce:_ee mg» we :ewpewm pcmweewm we ewepm mg» moms: emeemwwe