OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. THE ROLE OF MESSENGER RNA IN NASCENT PEPTIDE CHAIN ACCUMULATIONS By Wiliiam G. Chaney A THESIS Submitted to Michigan State University in partial fulfi11ment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1979 ABSTRACT THE ROLE OF MESSENGER RNA IN NASCENT PEPTIDE CHAIN ACCUMULATIONS By William G. Chaney An investigation of the origin of the nonuniform size distribution of rabbit globin nascent peptide chains was performed. Uniformly labeled globin nascent chains were isolated from a rabbit reticulocyte lysate cell-free protein synthesizing system. Analysis of the sizes of the nascent chains by gel exclusion chromatography under denaturing conditions was performed. Accumulations of nascent chains of discrete sizes were observed, as reported previously for nascent chains isolated from whole reticulocyte incubations (Protzel and Morris, 1972). Alteration of the relative concentrations of the components of the lysate protein biosynthetic machinery did not change the gel elution profile. A wheat embryo-derived cell-free messenger RNA-dependent protein synthesizing system was prepared. Globin mRNA was isolated from rabbit reticulocytes and added to the cell-free system. Gel chromatographic analysis of the labeled nascent chains purified from the rabbit globin mRNA-directed wheat embryo-derived cell-free protein synthesizing system demonstrated nascent chain accumulations of the same sizes as observed in nascent chains isolated from rabbit reticulocytes. The origin of globin nascent chain accumulations was seen to reside, therefore, in some prOperty of the globin mRNA itself, and not in the William G. Chaney protein biosynthetic machinery within which the globin mRNA is under- going translation. Infection of rifampicin treated g, gglj_with the RNA bacteriophage, M52, was found to yield, fifty minutes after infection, predominantly M52 coat protein biosynthesis. Nascent chains purified from M52 infected, rifampicin treated g, gglj_fifty minutes after infection will contain predominantly M52 coat protein nascent chains. Gel chromato- graphic analysis of nascent chains purified from M52 infected, rifampicin treated g, 9911 gave evidence for the existence of nascent chain accumu- lations during M52 coat protein biosynthesis. jg_!jvg, A comparison was made between a model of the M52 RNA coat protein genome secondary structure and the positions on the genome where ribosomes would reside in a population of mRNA molecules for greater relative lengths of time to generate the observed nascent chain accumulations. The data presented in this thesis support the hypothesis that the nascent chain accumulations are due to regions of mRNA secondary structure impeding the rate of ribosome movement during the process of peptide chain elongation. ACKNOWLEDGMENTS I would like to thank Dr. Allan Morris for his guidance and help during the course of my graduate study, and Drs. F. Rottman, R. 2 Patterson, and N. Smith for serving on my graduate advisory committee. Special thanks go to my wife, Linda, for many hours of nimble- fingered clerical assistance and moral support. ii TABLE OF CONTENTS Page LIST OF TABLES ......................... V LIST OF FIGURES ........................ vi INTRODUCTION .......................... 1 Protein Biosynthesis ................... l Hemoglobin Synthesis in the Rabbit Reticulocyte ...... 2 Control of Hemoglobin Synthesis by Hemin ......... 3 Protein Biosynthesis in RNA BacteriOphage Infected E. coli ....................... 4 Structure of Nucleic Acid Polymers ............ 9 Transfer RNA ....................... lO Ribosomal RNA ....................... l2 SS Ribosomal RNA ................... 13 165 and 185 Ribosomal RNA .............. l4 BacteriOphage RNA ..................... l7 BacteriOphage T7 RNA ................... l8 Eukaryotic Messenger RNA ................. l9 Globin Messenger RNA ................... 20 Nascent Chain Nonuniformities in Rabbot Reticulocytes . . . 22 MATERIALS ........................... 25 ‘ METHODS ............................ 27 Preparation of Rabbit Reticulocytes ............ 27 Preparation of Reticulocyte Lysate ............ 28 Preparation of Wheat Embryo Cell-Free Protein Synthesizing System ................. 28 Determination of the Rate of Protein Synthesis ...... 29 Growth Condition of E, coli ................ 30 Preparation of M52 StocE ................. 33 Purification of Peptidyl-tRNA ............... 33 Recrystallization of Guanidine-HCl ............ 37 Analysis of Peptide Sizes ................. 40 Isolation of Rabbit Globin mRNA .............. 41 Preparation of Aminoacyl-tRNA Synthetases from Rabbit Reticulocytes .................... 42 Preparation of a Salt Nash of Reticulocyte Ribosomes . . . 45 Ammonium Sulfate Fractionation of a Reticulocyte Post- ribosomal Supernatant Fraction ............ 45 Isolation of Rabbit Reticulocyte tRNA ........... 46 Isolation of Rabbit Liver tRNA .............. 49 Determination of Leucine Accepting Capability of tRNA . . . 50 Treatment of tRNA with Periodate ............. Sl Formaldehyde Treatment of Globin mRNA ........... 52 Cyanogen Bromide Treatment of M52 Coat Protein ...... 53 Separation of Alpha and Beta-Globin ............ 53 Cyanogen Bromide Treatment of Rabbit Alpha and Beta- Globin ........................ 54 Determination of Distribution Coefficients ........ 54 Preparation of Proteins for 505 Gel ElectrOphoresis . . . . 57 Polyacrylamide Gel ElectrOphoresis of Proteins ...... 58 RESULTS ............................ 6O Reticulocyte Lysate Cell-Free System ........... 60 Steady State Labeling ................... 6l Addition of Reticulocyte tRNA to the Reticulocyte Lysate . 65 Characterization of Rabbit Liver tRNA ........... 67 Addition of Rabbit Liver tRNA to the Reticulocyte Lysate . 72 Addition of Periodate Treated tRNA to the Reticulocyte Lysate ........................ 76 Addition of Salt Nash Factors to the Reticulocyte Lysate . 76 Addition of Ammonium Sulfate Precipitated Factors to the Reticulocyte Lysate ................. 82 Inhibition of Protein Biosynthesis by Edeine ....... 82 Wheat Embryo Cell-Free System ............... 88 Attempts to Modify Globin mRNA In Vitro .......... 108 The Effect of Spermidine Upon thE Nascent Chain Size Distribution ..................... llZ Heat Treatment at 65° of Globin mRNA ........... llS DMSO Treatment of Globin mRNA ............... llB Formaldehyde Treatment of Globin mRNA ........... lZl Bacteriophage M52 Protein Synthesis ............ l30 Analysis of Nascent Chain Sizes from M52 Infected E. coli . l37 Elution Profile of Nascent Chains from Uninfected E} coli . T45 Base Lability of the E, coli Nascent Chains ........ 146 Test of the Bio-Gel Column with Alpha- and Beta-Globin Derived Peptides ................... l49 Isolation of M52 Coat Protein Derived Peptides ...... l57 Calibration of the Bio-Gel a Column with M52 Coat Protein Derived Peptide Markers ............... 165 DISCUSSION ........................... T74 BIBLIOGRAPHY .......................... 200 iv Table 10 ll 12 LIST OF TABLES Final concentrations of components added to the reticulocyte lysate incubation mixture ......... Final concentrations of components added to the wheat embryo cell-free incubations .............. Contents of MTPA media ................. Composition of st0pping buffer ............. Incubation mixture for the determination of leucine accepting capability of tRNA .............. Treatment of rabbit liver tRNA to determine leucine acceptance capability .................. Leucine acceptance capability of sodium periodate treated rabbit liver tRNA ................ Distribution coefficients of nascent M52 coat protein chains from nine separate experiments using leucine, aspartic acid, and asparagine to radioactively label nascent chains ..................... A comparison of the distribution coefficients of a- and B-globin peptide markers obtained by Protzel (1973) and in Figures 36 and 37 ................ Data derived from Figures 4l and 42 which have been used to calibrate the Bio-Gel A 0.5 column for M52 coat protein derived peptides ................ Molecular weights calculated for M52 coat protein nascent chain accumulations of Table 9 using the rela- tionship expressed in Figure 43 ............. Amino acid lengths of M52 coat protein nascent chain accumulations calculated from the data in Table ll Page 28 30 31 32 51 7O 72 145 157 T70 173 189 Figure 01-th OS 10 11 12 13 14 15 16 LIST OF FIGURES The nucleotide sequence and pr0posed secondary structure of the bacteriophage M52 coat protein cistron ......................... Desalting step of peptidyl-tRNA purification ...... Elution of peptidyl-tRNA from DEAE-cellulose ...... Oligo(dT)-cellulose chromatography of globin mRNA . . . . Elution profile of rabbit reticulocyte tRNA from DEAE-cellulose ..................... Elution of globin from CM-cellulose ........... Steady state labeling of ribosomes ........... The effect of reticulocyte tRNA upon nascent chain accumulations ...................... The effect of rabbit liver tRNA upon nascent chain accumulations ...................... The effect of periodate-treated rabbit liver tRNA upon nascent chain accumulations ............... The effect of salt wash factors upon nascent chain accumulations ...................... The effect of (NH ) SO precipitated proteins upon nascent chain accflmalagi Inhibition of protein biosynthesis by edeine ...... The effects of edeine upon nascent chain accumulations Elution profile of nascent chains from a lysate con- taining edeine ..................... Effect of mRNA concentration upon protein synthesis in the wheat embryo cell-free system .......... vi ODS ............... Page 36 39 44 48 56 64 69 75 78 81 84 87 90 92 95 Figure 17 18 19 20 21 22 23 24 25 26 27 28 29 3O 31 32 Protein synthesis as a function of Mg(Ac) concen- tration in the wheat embryo cell-free sys em ...... Incorporation of 3H-leucine into protein as a function of time in the wheat embryo cell-free system ......................... Elution profile of nascent chains from the globin mRNA directed wheat embryo cell-free system ....... Elution profile of nascent chains from the globin mRNA directed wheat germ cell-free system ........ Analysis of the soluble products of the globin mRNA directed wheat germ cell-free system .......... Elution profile of polysomal material from the globin mRNA directed wheat embryo cell-free system ....... Elution profile of polysomal material from the reticulocyte lysate ................... Effect of Spermidine upon the nascent chain elution pattern in the globin mRNA directed wheat embryo cell- free system ....................... Elution profile from the heat treated globin mRNA directed wheat embryo cell-free system ......... Elution profile from the DMSO treated globin mRNA directed wheat embryo cell-free system ......... Elution profile from the formaldehyde treated globin mRNA directed wheat embryo cell-free system ....... Effect of formaldehyde treatment Of globin mRNA Upon the elution profile of nascent chains from the wheat embryo cell-free system ................. Bacteriophage M52 inducted protein synthesis in rifampicin treated E, coli ............... A SDS polyacrylamide gel analysis of the products of M52 infected, rifampicin treated E,coli .......... Elution profile of 3H-leucine labeled nascent chains from M52 infected, rifampicin treated E, coli ...... Elution profile of nascent chains isolated from M52 infected rifampicin treated E, coli 30 minutes after infection ........................ vii Page 97 99 102 105 111 114 117 126 128 139 142 Figure 33 34 35 36 37 38 39 4O 41 42 43 44 45 Elution profile of 3H-aspartic acid labeled nascent chains from M52 infected, rifampicin treated E, coli Elution profile of nascent chains from uninfected E, coli ......................... The possible peptides derived from a- and B-globin by cyanogen bromide treatment .............. Elution profile of cyanogen bromide treated B-globin ........................ Elution profile of cyanogen bromide treated a- and B-globin ........................ The possible peptides derived from M52 coat protein by cyanogen bromide treatment .............. Elution profile of M52 coat protein ...... .. . . . . Chromatographic analysis of the peptides derived by treatment of M52 coat protein with cyanogen bromide . . . Chromatographic analysis of M52 coat protein and cyanogen bromide derived peptides ............ Bio-Gel chromatography of M52 coat protein and coat protein cyanogen bromide derived peptide markers . . . . A graph of the relationship between Kdv3 and (mw)0-555 for M52 derived peptides ................ The positions to which nascent chain accumulations correspond on the M52 coat protein cistron nucleotide sequence ........................ An alternate structure for a portion of the M52 coat protein cistron ..................... viii Page 144 148 152 154 156 160 162 164 167 169 195 INTRODUCTION Protein Biosynthesis Messenger RNA (mRNA) contains information in its nucleotide se- quence for the amino acid sequence of the protein to be assembled. The amino acids are initially bound to a transfer RNA (tRNA) molecule capable of interpreting the genetic code within an mRNA sequence and synthesis takes place upon a cellular organelle known as a ribosome. The assembly of amino acids into proteins takes place in a step-wise manner from the N-terminal to the C-terminal end of the protein (Bishop g5_gl,, 1960; Dintzis, 1961). The ribosome moves along the mRNA in a 5' to 3' direction (Ochoa, 1965) and each translating ribo- some contains only one growing peptide chain (Warner and Rich, 1964). The rate at which a protein is produced by a mRNA molecule may be affected by the rate of initiation of protein synthesis, the rate of peptide bond formation, the rate at which the completed chains are released from the ribosomes, and the amount of mRNA present. The stability of the mRNA undergoing translation and the rate at which a protein is degraded by intracellular proteases are factors which will affect the amount of protein present in a cell (Schimke, 1973; Nomura gt_gl,, 1974; Weissbach and Ochoa, 1976; Weissbach and Pestka, 1977). 2 Hemoglobin Synthesis in the Rabbit Reticulocyte Reticulocytes are immature erythrocytes which are still synthe— sizing protein. In mammals the nuclei of the reticulocytes have been extruded from the cell. Greater than 95% of the protein synthesis in rabbit reticulocytes is hemoglobin synthesis (Woodward g5_gl,, 1972). Rabbit hemoglobin contains two different proteins, a- and B-globin, which are present in a tetramer (aZBZ). Alpha globin has been reported to be synthesized at a slightly higher rate than B-globin in reticulo- cyte incubations (Tavill gE_§l,, 1968) and the ribosomes of rabbit reti- culocytes have been shown to contain approximately equal numbers of a- and B-globin nascent chains (Hunt gE_gl,, 1968, Lodish, 1971, Protzel and Morris, 1973). Beta globin is synthesized on larger poly- somes than a-globin (Hunt gE’gl,, 1968), and the amount of a-globin mRNA in a reticulocyte is larger than the amount of B-globin mRNA in a reticulocyte (Lodish, 1971). The average translation rates of both a- and B-globin have been determined to be equal, about 200 seconds per chain synthesized at 25° (Lodish and Jacobson, 1972; Hunt, 1974). A model for hemoglobin biosynthesis, which incorporates the obser- vations described above, has been described by Lodish (1976). Beta- globin mRNA, having a higher affinity for ribosomes than a-globin mRNA, undergoes initiation of protein synthesis at a higher rate than the initiation rate for a-globin mRNA. The higher rate of B-globin initia- tion would explain the increased sizes observed for polysomes synthe- sizing B-globin (Hunt gE_gl,, 1968). The higher concentration of a- globin mRNA relative to B-globin mRNA, reported to be present in the rabbit reticulocyte, has been proposed to balance the higher rate of 3 initiation of B-globin synthesis in order to yield approximately equal numbers of a- and B-globin nascent peptides (Lodish, 1971, 1976). Control of Hemoglobin Synthesis by Hemin Early studies of protein synthesis in isolated rabbit reticulo- cytes have demonstrated a requirement for added ferrous ion (Kruh and Borsook, 1956). Without added Fe++ the rate of incorporation of radio- active amino acids into protein greatly decreases after a few minutes. Each globin has a heme group bound to it which contains iron, suggesting the existence of a mechanism of coordinate control of globin and heme biosynthesis. The requirement for Fe++ has been found to be replaceable by hemin (Waxman and Rabinovitz, 1966; Bruns and London, 1965), support- ing the conclusion that the stimulatory effect of iron is through increased heme biosynthesis. However, binding of heme to nascent chains could not be demonstrated, providing evidence that heme binds to globin after globin biosynthesis upon reticulocyte ribosomes (Morris and Liang, 1968). Cell-free protein synthesizing systems prepared from the reticulo- cyte lysate have also been shown to have a requirement for added hemin. If hemin is not present in reticulocyte lysate incubations, an inhibitor of the initiation step of protein synthesis rapidly forms at 37° (Zucker and Schulman, 1968; Adamson gt_gl,, 1968). The formation of a protein synthesis inhibitor in_gj§rg_is thought to occur by the same mechanism that yields the inhibition of protein synthesis in iron deprived cells in whole cell incubations. This hemin controlled repressor (HCR) is a protein with a molecular weight of about 400,000 and is formed from a precursor of the same molecular weight (Adamson 3511,1972). 4 Inhibition of protein synthesis by HCR is thought to be due to phOSphorylation of a protein synthesis initiation factor, eIF-2 (Levin gE_gl,, 1975, 1976; Balkow g3_gl,, 1975; Farrell gE_§1,, 1977). Addi- tion of purified eIF—2 to hemin deprived reticulocyte lysate incuba- tion mixtures has been shown to relieve the incubation mixtures from the inhibition of protein synthesis initiation (Kaempfer gE_gl,, 1974; Clemens g3_gl,, 1977). The phosphorylation of eIF-2 has been reported to be performed by a cyclic AMP independent protein kinase which, in turn, may be activated by phosphorylation by a cyclic AMP dependent protein kinase (Levin gE_§1,, 1976; Farrell gE_gl,, 1977; Kramer gE_gl, 1976). Hemin has been demonstrated to prevent the binding of cyclic AMP to the cyclic AMP dependent protein kinase in a manner which is thought to control the activity of the cyclic AMP dependent protein kinase (Datta §E_gl,, 1978). The inhibition of protein synthesis initiation by HCR has also been shown to affect the initiation of non- globin protein synthesis in rabbit reticulocyte lysate incubation mixtures (Lodish and Desalu, 1973). Hemin dependent mechanisms of control of the initiation of protein synthesis have also been reported to exist in HeLa cells and in Krebs II ascites tumor cells (Beuzard g}_gl,, 1973; Weber 3} 91,, 1975). Protein Biosynthesis in RNA BacteriOphage Infected E. coli A class of bacteriOphages containing RNA as their genome has been described by Loeb and Zinder (1961). This RNA is capable of acting as a mRNA molecule jg_vivo and ig_vitro. Two classes of RNA phages have been found. One class consists of the closely related phages, M52, R17, f2, M12, u2 and the other class, the serologically distinct 5 phage 08. All these phages are specific for "male" E, gglj_since they enter the cell by adsorbing to the F pili during infection. A collection of reviews summarizes much of the knowledge available about these bacteriOphages (Zinder, 1975). The genome of the M52 bacteriOphage contains 3569 nucleotides which include the genetic information for three viral coded proteins synthesized by infected E, gglj_(Fiers gE_gl,, 1976). They are, in order from the 5' end of the viral genome, the maturation protein, the coat protein, and the replicase protein. The replicase protein has been shown to form an RNA-dependent RNA polymerase with three native E, gglj_proteins, the elongation factors Tu and Ts, and the 51 protein of the small ribosomal subunit (Federoff, 1975). The RNA- dependent RNA polymerase is responsible for replication of the bacterio- phage genome. Synthesis of replicase protein is thought to require prior translation of the coat protein RNA sequence. Bacteriophage mutants containing nonsense mutations (which cause premature termination of protein synthesis) near the beginning of the coat protein messenger RNA sequence have been shown to produce only a small amount of repli- case protein when the bacteriophage RNA is added to an E, ggljfderived cell-free protein synthesizing system. BacteriOphage RNA containing nonsense mutations in the last half of the coat protein RNA have been found to synthesize normal amounts of replicase protein under the same jg_!1339_conditions (Tooze and Weber, 1967; Capecchi, 1967; Engelhardt, 1967). Production of intact, functional coat protein does not seem to be required for replicase synthesis, only translation of the mRNA sequence 6 which contains the genetic information for the N-terminal half of the coat protein. A change of conformation has been proposed to occur when a ribosome traverses the mRNA sequence which codes for the N- terminal half of the coat protein. The change of conformation is thought to expose the initiation region of the M52 replicase protein, which has been concluded to exist in intact, untranslated M52 RNA associated with part of the 5' half of the M52 coat protein cistron in a structure which prevents initiation of replicase protein synthesis at this site (Lodish and Robertson, 1969). Studies of the nucleotide sequence of the M52 genome have demon- strated a region in the coat protein cistron which could undergo intra- molecular base pairing with the regions at the start of the replicase subunit cistron (Min Jou gE_gl,, 1972). A proposed structure of the coat protein genome is shown in Figure 1. The structure which may be formed between the M52 coat protein cistron RNA and the region bearing the initiation codon of the replicase cistron would be forced apart during translation of the M52 coat protein mRNA region by a ribosome. The replicase protein synthesis initiation region would then be exposed. Figure 1, therefore, provides a structural explanation for the dependence of replicase protein synthesis upon translation of the 5' end of the M52 coat protein mRNA sequence ig_gi§gg, The ratio of coat protein synthesis to replicase protein synthesis changes during the course of infection of E, ggli_with an RNA bacterio- phage. Early in infection the molar ratio of coat protein synthesis to replicase protein synthesis is 6:1. Late in the phage reproductive cycle the ratio of coat protein synthesis to replicase protein synthesis becomes greater than 30:1 (Kozak and Nathans, 1972). An explanation .cocpmwo cwmuoca “moo mm: mmmcaowcmuomn mg» mo meaguscgm xgmucoumm ummoaoca use oucmzcmm muwuompozc one .F mczmwm O . / 4 99 o //L _ 4 I99 0 O o o Q0//0/? ucom 390 9:»..95 H 0 “Info .0 use “on” ”Vinson” N / . . . H UQ/QQ Ono \ ‘8 0350:: H w i o O col/0010 O¥/. N S9. \00 v o J“ Vlorf/ O . 883.2032 o co \ wwv v w x66 8...» 2.5 a. v... - vow co v 09/ 01w of. 88... 8.5:... 0 «ouoagoo : cocw/Woo .< ems, \ Lao <3...m.i.:..?.. oo: 00333044 1‘ .A . .2220 J/oom/ no: mo. ”.3... _ _ .«wwwwww?w._.ww_<«wwp “Page; mo uuweem och .m «came. 75 1.5- 1.0 10— 0.5” 2.01 x was Figure 9 76 which received no additional tRNA. Therefore, rabbit liver tRNA as well as rabbit reticulocyte tRNA were concluded to have no effect upon the nascent globin peptide accumulations. Addition of Periodate Treated tRNA to the Reticulocyte Lysate A lysate incubation was also performed with the addition of 10.0 A260 units of sodium periodate treated rabbit liver tRNA added to one m1 of the lysate incubation mixture. The periodate treated tRNA sample had been shown to have no leucine accepting capability, as reported above. Nascent chains were labeled with 3 H-tryptophan, purified from the incubation mixture, and analyzed by Bio-Gel chromatography as described in Methods. Comparison of the elution profile of Figure 10 with the 14C-tryptophan labeled profile of Figure 8 showed no signifi- cant alteration in the nascent chain elution profile. Addition of a tRNA population incapable of participating in protein synthesis also did not affect the nascent chain accumulation pattern. Addition of Salt Wash Factors to the Reticulocyte Lysate The inability of tRNA preparations from rabbit reticulocytes or rabbit liver to alter the nascent chain size distribution elution profile led to attempts to determine if the nascent chain size accumu- lations were related to the limitation of other components of the reticulocyte lysate cell-free protein biosynthetic machinery. Treat- ment of rabbit reticulocyte ribosomes with high salt concentrations and of rabbit reticulocyte postribosomal supernatants with 70% (NH4)2504 have been reported to yield preparations containing protein synthesis initiation and elongation factors (Woodley e3.el,, 1974; Hardesty eE_ al., 1971). Different fractions of the reticulocyte lysate were 77 .mcowum_353oom ewmgo ucmomm: cog: .— genome umpmmcu-mpeeowcma mo uomeem web .0. deemec 78 0.. op «gnaw; to. .540 0.0 d 79 prepared for use in an analogous manner to the experiments involving the addition of tRNA to the reticulocyte lysate incubation mixture. If the nascent chain Size accumulations were due to the limitation of a component present in such a preparation, addition of the preparation to the lysate incubation mixture would be expected to reduce the heights of the peaks of the accumulations in the Bio-Gel elution profile. Treatment of polysomes with 0.5 M KCl had been reported to release protein factors involved in the initiation of protein biosynthesis (Woodley eE_el,, 1974). Acting upon the premise that the 0.5 M KCl wash preparations might contain factors associated with nascent chain accumulations, reticulocyte lysate polysomes were washed with 0.5 M KCl as described in Methods. The material eluting from the polysomes was concentrated and added to the lysate cell-free system to give a concentration in the incubation mixture three times that present I originally. Nascent chains were labeled with 3H-tryptOphan, purified, and analyzed upon Bio-Gel A 0.5 m under denaturing conditions with an internal standard consisting of 14C-tryptophan labeled nascent chains that had been co-purified from a lysate which received no additions. As shown in Figure 11, the results of the experiment described above failed to demonstrate any change in the nascent globin peptide elution profile upon addition to the lysate cell-free system of the factors eluted from the lysate polysomes by 0.5 M_KCl. If such a limiting factor did exist in the 0.5 M_KCl wash of reticulocyte lysate 3H-labeled nascent chains would ribosomes, the elution profile of the have shown a decrease in the heights of the nascent chain size accumu- lations. 80 .mcowumF353oom cemco ucmommc cog: mcouomw 2mm: upmm mo Humvee one ... de=m_. 81 (—-°-—)3.Ol x WdO 0,, N T 1 N (+),.OI X WdO H$2 l.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 ”00 0| 82 Addition of Ammonium Sulfate Precipitated FactOrs to the Reticulocyte Lysate Adjustment of a reticulocyte lysate postribosomal supernatant to 70% (NH4)2504 had been reported to precipitate protein factors involved in the elongation steps of protein synthesis (Hardesty eE_el,, 1971). A reticulocyte lysate postribosomal supernatant was adjusted to 70% (NH4)2504 at 0° by the Slow addition of powdered (NH4)2504 at 0° as described in Methods. The material precipitated by 70% (NH4)250.4 was added to a lysate incubation mixture to bring the final concentration of the prepared factors to three times that normally present. Nascent chains were uniformly labeled with 3 H-tryptophan. A control incubation with 14C-tryptophan was prepared for use as an internal standard. Ribo- somes from both incubations were mixed and the nascent chains purified as described previously in Methods. Gel chromatographic analysis of the purified nascent chain popu- lations yielded elution profiles for the experimental 3H-labeled nascent 14C-labeled nascent chains. No alterations in chains and the control the nascent chain accumulation profile were induced by the addition of the factors precipitated by 70% (NH4)2504 (Figure 12). The discon- tinuity of the elution profile was due to the failure of a fraction collector. From the graphs in Figure 12, no evidence for the presence of a factor or factors in the material precipitated by 70% (NH4)2504 which could be related to the origin of the nascent globin chain accumulations could be found. Inhibition of Protein Biosynthesis by Edeine The preceding experiments failed to provide evidence for the involvement of a factor or factors of the reticulocyte lysate cell-free 83 .mcowuepzsauoe cease ucmomec cog: mcwmuoea uopeuwapomca eommfiezz. co “emcee we» .N_ oesmwe 84 X “CM 0171 01 NP «Laure ex 0.0 to _ _ _ q 85 protein biosynthetic system in the accumulation of nascent o- and 8- globin peptides of discrete Sizes during protein biosynthesis. How- ever, these iesults did not definitively disprove the hypothesis that such factors were associated with the accumulations observed in the nascent globin chains of rabbit reticulocytes. Such components of the reticulocyte lysate could exist and be either not purified by the isolation procedures used or destroyed by these isolation procedures. Therefore, a separate and entirely independent approach was taken to examine the possibility that the nascent chain accumulations observed in rabbit reticulocytes were due to a stoichiometrically limiting factor or factors of peptide chain elongation. The antibiotic, edeine, had been reported to inhibit protein bio- synthesis specifically at the step of initiation (Obrig eE_el,, 1971). The effects of increased concentrations of edeine upon the rate of protein biosynthesis in the reticulocyte lysate incubation mixture were determined. The results of this experiment are shown in Figure 13. An edeine concentration of 19 pM_was found to inhibit protein bio- synthesis to about 25% of its original rate. Since edeine reportedly inhibits the initiation step of protein biosynthesis, the rate of nascent chain elongation should be unchanged. The number of ribosomes involved in peptide chain elongation in an incubation with added edeine would, therefore, be expected to be one-fourth of the number of active ribosomes found in an uninhibited incubation mixture. The reduction in number of actively translating ribosomes would have the effect of increasing by four times the concentrations of the individual components which were involved in nascent peptide chain elongation (elongation factors, tRNA, etc.) relative to the concentration of the 86 Figure 13. Inhibition of protein biosynthesis by edeine. 87 100 INHIBITION PERCENT 50 l l l J I l o ’ 20 4o EDEINE CONCENTRATION In") Figure 13 60 88 active ribosomes. The nascent chain accumulations which had been observed would be expected to be decreased or eliminated if they were indeed due to a limiting concentration of a factor or factors involved in peptide chain elongation. Nascent globin chains were labeled with 3 H-tryptophan in a reti- culocyte lysate incubation mixture which contained 19 pM_edeine. Ribosomes isolated from the incubation containing edeine were mixed with ribosomes from a lysate incubation mixture to which no edeine was added. In the control incubation nascent globin chains were labeled with ‘4 C-tryptophan. The nascent chains were isolated and Bio-Gel A 0.5 m chromatographic analysis performed. The results from two separate experiments performed in such a manner, shown in Figure 14 and 15, indicated that no alteration was induced in the nascent globin chain accumulation pattern from the lysate incubation mixture in which protein synthesis was inhibited by the addition of 19 pM_edeine. The results obtained by the addition of edeine to the cell-free lysate incubation mixtures were in agreement with the results obtained in the attempts to purify a putative factor or factors from the reti- culocyte lysate cell-free protein synthesizing cell-free system. Neither approach was able to provide evidence for the existence of such a factor. It was, therefore, concluded that such a factor did not exist and that the origin of the nascent globin chain accumulations originated in some other aspect of o- and B-globin biosynthesis. Wheat Embryg Cell-Free System The inability of the experiments described in the preceding section to demonstrate a factor or factors in the reticulocyte lysate, which 89 .meompepzssooe cwmco pcmummc cog: mewmum mo muommem one .e. mesmee 90 no. 0| X WdG H )2 91 .mcwmem mcecwmpcoo ovum»? m Eoce meeego pcwome: co m_wwoeq cowu:_m .m. desm_. 92 —"’ 2-01 1‘ We“) 0 in ID '53- 2 o’ ' I °_ 2.Ol x lNdCI H N d Figure 15 93 could be the origin of the nascent globin peptide accumulations, led to an attempt to determine if these accumulations were associated with translation of globin mRNA by the protein biosynthetic machinery of the reticulocyte lysate, or if Similar size accumulations were present in nascent chains derived from globin mRNA directed protein biosyn- thesis in an entirely different cell-free system. The wheat embryo cell-free system was chosen for these experiments because of its dependence upon added mRNA for protein synthesis and its phylogenetic distance from rabbits. Globin mRNA was prepared from rabbit reticulocytes by the method of Aviv and Leder (1972). The wheat embryo cell-free system was prepared by the method of Marcus eE_el, (1975). The incorporation of 3H-leucine into protein was dependent upon added globin mRNA (Figure 16). The rate of protein synthesis showed an optimum at a concentration of 3.6 mMMg(Ac)2 in Figure 17. An incubation of globin mRNA at a concen- 3 tration of 8 pg/ml gave a linear rate of incorporation of H-leucine into protein (Figure 18). Nascent chains were labeled with 3 H-tryptophan for 20 minutes in an incubation mixture of 5.6 ml containing 6.5 pg globin mRNA per ml. Protein synthesis was halted and ribosomes isolated as described in Methods. The ribosomes were then mixed with ribosomes isolated by centrifugation from a reticulocyte lysate in which the nascent chains had been uniformly labeled with ‘4 C-tryptophan. The combined nascent chains were purified and Bio-Gel A 0.5 m chromatographic analysis was performed. The analysis of the nascent chains purified from the wheat embryo incubations indicated that nascent chain accumulations were also present 94 Figure 16. Effect of mRNA concentration upon protein synthesis in the wheat embryo cell-free system. OP»! 3: no" .5 2 95 4 6 a 10 GLOBIN m RNAng/mll Figure 16 96 Figure 17. Protein synthesis as a function of Mg(Ac)2 concentration in the wheat embryo cell-free system. 0PM 1:10"3 A N 97 I l 2 3 Figure 17 98 Figure 18. Incorporation of 3H-leucine into protein as a function of time in the wheat embryo cell-free system. CPM x IO 99 l l l IO 1 20 30 Time (min) Figure 18 4O 100 in the wheat embryo cell-free system during globin mRNA dependent protein synthesis (Figure 19). Accumulations of nascent chains at a Kd greater than 0.4 (> 80 amino acids in length) were correlated between the lysate and wheat embryo systems. The relative magnitude of the nascent chains at distribution coefficients less than 0.4 was decreased, probably due to premature termination in the wheat embryo cell-free system similar to that described by Roberts eE_el, (1973). Previous experiments with nascent chains purified from the reti- culocyte lysate cell-free system had shown that the relative heights of the peaks in the gel elution profiles could vary between two elution profiles derived from two separate Bio-Gel chromatographic analyses of the same nascent globin chain sample. Hence, addition of 14C-tryptophan labeled polysomes as an internal standard was employed to correct for any technical difficulties during the nascent chain purification and column chromatography. Most experiments involving the wheat embryo cell-free system were performed using only a single 3H-amino acid as the radioactive label. In all of these analyses the presence of nascent globin chain accumulations of the same sizes as those observed in the reticulocyte lysate and whole cell incubations was quite evident. Not until the experiment shown in Figure 19 using 14C-tryptOphan labeled nascent chains from the rabbit reticulocyte lysate as an internal control was performed was the presence of the decrease in the nascent chain profiles at Kd values less than 0.4 adequately demonstrated. The decrease had been observed in the Single label experiments, but evaluation of the differences in magnitude was felt to be unreliable. 101 .EOpmAm «mew -Fpmu ozgaew poms: umpumcwu wsmu mmnwuama mpawmmoa mgh .mm scene; 152 mm 2.5: p «-36 _ . mo. .p 78-0 . .1 . 1E 250.66 0 «n3; 2 «-mo- — _ q . Tumol .m _ I. q no _ T L. 1“ 2.849% o 3.32 z 153 .cwnopmum caucus» muwsogn camocmxu mo mpwmoca cowuapm .em mesmee 154 mm «eased 3. who 155 .cwno—mim use -5 cavemen caveats camocmzu to m_weoca co_uspm .mm weaned 156 0.. mm «gamed 3. no 0.0 157 in Table 9. Four peptides, whole B-globin, B-CB-l, B-CB-Z, and a-CB-l all had experimentally determined Kd values which were within 0.01 Kd units of the Kd values determined for these peptides by Protzel (1973). The close agreement of the experimentally determined Kd values was evidence that the linear relationship determined by Protzel and Morris 1/3 and (molecular weight)0'555 (1974) and Fish gE_gl, (1969) between Kd was indeed valid for the chromatographic analyses performed in this thesis. Table 9. A comparison of the distribution coefficients of a- and B- globin peptide markers obtained by Protzel (1973) and in Figures 36 and 37. 'Protzel Figure 11 Figure 36 Figure 37 B-globin (146) 0.22 0.21 B-CB-Z (91) 0.33 0.33 B-CB-l (55) 0.52 0.53 0.52 a-CB-Z 0.30 (0.30) a-CB-l (32) 0.63 0.64 Isolation of M52 Coat Protein Derived Peptides The Bio-Gel A 0.5 m column, used for the analysis of nascent chain accumulation patterns, was calibrated using radioactively labeled M52 coat protein and peptides derived from M52 coat protein by treatment of a radioactively labeled, coat protein fraction with cyanogen bromide. The calibration with coat protein peptides was performed for the purpose of determining the molecular weights of M52 coat protein 158 nascent peptides. The possible peptide fragments which may be derived from cyanogen bromide treatment of M52 coat protein are outlined in Figure 38. The fraction, which did not bind to DEAE-cellulose during the purification of 3H-leucine nascent peptide chains from M52 infected E, 5911, was used as a source of radioactively labeled M52 coat protein since M52 coat protein had been shown to be the major (> 80%) radioactively labeled protein product of the E, gglj_incuba- tions. Analysis of the M52 coat protein sample as described above by Bio-Gel A 0.5 m chromatography under denaturing conditions demonstrated a single peak of radioactivity with a Kd of 0.25 (Figure 39). The M52 coat protein sample was used for preparation of radio- actively labeled peptides by cyanogen bromide treatment since MSZ coat protein was the only protein which was radioactively labeled as shown above. The products of cyanogen bromide treated M52 coat protein were analyzed by Bio-Gel A 0.5 m chromatography. The elution profile obtained from chromatographic analysis of a preparation of cyanogen bromide treated coat protein is shown in Figure 40. Three peaks of radioactivity were seen. All were of a Kd value higher than than that of unreacted coat protein (smaller molecular weights). All the coat protein in this sample had, therefore, reacted with cyanogen bromide in at least one of the two methionines. The peak at Kd = 0.557 clearly corresponded to a peptide 41 amino acids long. The accumu- lations at Kd = 0.716 were from peptides 20 and 21 amino acids long. The peak of radioactivity at Kd = 0.345 was assigned a length of 88 amino acids for two reasons: 1) the size of a peptide 88 amino acids long corresponded to the molecular weight calculated for this Kd value from the data of Protzel and Morris (l974) using a- and B-globin 159 Figure 38. The possible peptides derived from M52 coat protein by cyanogen bromide treatment. 160 MET MET 88 108 (129) (108) (88) (41) (20) (21) Figure 38 161 .cwmuoga umou Nmz mo mpwmogn =o_p:_m .mm ee=m_e 162 0.. mm ms=m_u 3 no 0.0 L :1. 2.0111de _.L '0 163 .wuwsoca :mmocezu :pwz cwmuoca pmoo mm: to pcmsammcp an um>wcwu mmuwpgma as» mo mwmxpmcm oeggmcmoumsocgu .oe weemwa 164 0.. 0e .e:u.m 3. 0.0 00 r- (I) If) ‘3' 1‘) ‘31 3.011: was 165 peptide standards, and 2) in order to generate peptides of 41 amino acids long (Kd = 0.557), the MS2 coat protein must be cleaved by cyanogen bromide only at amino acid position 88 and ggE_108. Because a single peak appeared at Kd = 0.345, only one of the two peptides (88 or 108 amino acids long) was possible. Since the labeled peptide which correSponded to 41 amino acids in length was present, the accumu- lation at Kd = 0.345 was concluded to be of peptides 88 amino acids long. Calibration of the Bio-Gel a Column with M52 Coat ProteinIDériVed Peptide Markers Peptide markers were prepared by cyanogen bromide treatment of 3H-leucine labeled MS2 coat protein as described in Methods. Labeled M52 coat protein (not treated with cyanogen bromide) was added to the cyanogen bromide treated M52 coat protein preparation and the peptides subjected to Bio-Gel A 0.5 m column chromatographic analysis after being dissolved in 0.4 ml of guanidine solution. Two separate analyses were performed and are shown in Figure 41 and 42. The Kd values were determined for each peak in Figures 41 and 42. The molecular weights were calculated from the amino acid composition of the peptides. The values obtained by such calculations are shown in Table 10. The values expressed in Table 10 were used to calculate the linear relationship between de3 and (Ml«l)o'555 for the M52 derived peptides using a Hewlett-Packard HP-65 calculator and the program for the solu- tion of the linear regression equation. The equation for the line calculated in such a manner was: (MN)0‘555 = 495.07 - (474.21)1wsmn mvwsoca cmGOmeU vcm :wmwocq pmou mm: mo mwm>chm ornamemopmsocgo ._e me:m.. 167 .e ecemwe 0.. 0.0 0.0 2.01 “1:10 1 (I? 168 .mcmxcme mvaama vm>wcmc muwsoca :mmocmxu cwmgocg pmou use cwmpoca umou Nmz mo mcamsmopmsoccu .moiovm .Ne ee=m_e 169 1.0 2-01 1: Hell) ' .. D. o o, o I 1 ‘0 <1- Ka Figure 42 170 Table 10. Data derived from Figure 41 and 42 which have been used to calibrate the Bio-Gel A 0.5 column for M32 coat protein derived peptides. 1/3 Molecular Kd (Kd) weight (hw20-555 Figure 41 Figure 42’ Figure 41 Figure 42 0.249 0.247 0.629 0.627 13,713 197.75 0.352 0.351 0.706 0.705 9,415 160.50 0.565 0.562 0.820 0.825 4,267 103.45 0.730 0.722 0.900 0.897 2,118 70.13 The equation on page 165 is expressed graphically in Figure 43. The line was a "good fit" for the data (r2 = 0.9936). The molecular weight ranges of the M52 coat protein mascent chain size accumulations was calculated from the Kd values and standard deviations expressed in Table 10 for M52 coat protein nascent chain size accumulations using this linear relationship. The molecular weight ranges in Table 11, therefore, represented the sizes of the nascent chain accumulations in the Bio-Gel elution profiles of M52 coat protein nascent chains and will be used in the Discussion section in a comparison of the regions of nascent M52 coat protein size accumu- lations and the proposed structure of the M52 genone. 171 .mmuwuama um>wgmu mm: Low mmm.o .35 2e m> ux :mmzumn awzmcowpmpmg ecu we cameo < .me mg=m_. 172 me 6236.. 88.0.32. com on. 00. Ayn . _ . mwgu sung mwflv Q“(‘51) 173 Table 11. Molecular weights calculated for M32 coat protein nascent chain accumulations of Table 9 using the relationship expressed in Figure 43. Peak Number Kda + 5.0. Molecular Heightc I 0.80 e 0.01b l,280-l,444 II 0.72 i 0.01 2,005-2,223 III 0.63 i 0.005 3,155-3,296 IV 0.52 i 0.01 4,869-5,26l V 0.39 i 0.01 7,907-8,495 VI 0.33‘: 0.005 9,973-10,337 VII 0.25 i 0.005 13,293-13,789 aData presented here summarize the results of nine analyses of the size distribution of nascent coat protein peptides. The distri- bution coefficient (K ) of each maximum in the Bio-Gel A 0.5 m elution profile is ingicated (see, for example, Figure 5). bDetected as a minor peak in five of the nine experiments. cThe range of molecular weights indicated by the standard deviation associated with each K value was calculated using the values obtained from a Bio-Ge A 0.5 m colume standardized with M52 coat protein and the peptides derived from M52 coat protein by cyanogen bromide cleavage (12). DISCUSSION The size distribution of nascent 0- and B-globin peptides in whole cell incubations of rabbit reticulocytes has been shown to be nonuniform (Protzel and Morris, 1974). Accumulations of discrete sizes were observed upon gel chromatographic fractionation of uniformly labeled nascent chains under denaturing conditions. The presence of size accumulations in the a- and B-globin nascent chain population isolated from rabbit reticulocytes implies that the rate of peptide chain elongation along the globin mRNA is not uniform. Some regions of the mRNA sequence are concluded to have a slower rate of peptide chain elongation than others. Since each ribosome involved in trans- lation has one nascent peptide chain attached to it, the accumulation of nascent chains of any length corresponds to the accumulation of ribosomes at a specific region along the mRNA being translated. The accumulation of ribosomes at Specific regions along the mRNA would be, therefore, the result of the lowered rate of ribosome movement along the mRNA in these regions (relative to other regions of that same mRNA). The results of Protzel and Morris (1974) demonstrated, there- fore, that the rate of peptide chain elongation in whole cell incuba- tions of rabbit a- and B-globin is not uniform. There are regions of the mRNA p0pulation which are translated relatively slowly compared to other regions of the mRNA. However, the origin of the difference in the relative rate of chain elogation was not investigated. The 174 175 purpose of the experiments described in this thesis was to investigate the origin of the nonuniform size distribution of rabbit globin nascent chains. Manipulation of the whole cell incubations in an attempt to analyze the origin of the nascent chain size accumulations would be quite difficult. The presence of the cellular plasma membrane prevents the easy addition of agents to the incubation mixture in attempts to affect the nascent chain size accumulations, as analyzed by elution of the nascent chain population from Bio-Gel A 0.5 m under denaturing conditions. Therefore, it was decided to isolate nascent chains from a cell-free protein synthesizing incubation mixture. Utilization of a cell-free protein synthesizing system has enabled experiments to be performed in which a component of the cell-free system involved in the process of nascent chain elongation was purified and then added to a cell-free incubation mixture in an attempt to determine if a stoichiometrically limiting amount of that component is the cause of the lowered relative chain elongation rates observed by gel chromato- graphic analysis of the nascent chain p0pulation for size accumulations. The rabbit reticulocyte lysate cell-free protein synthesizing system was chosen for these experiments for several reasons. First, synthesis of the same proteins studied by Protzel and Morris (1974), rabbit a- and B-globin, takes place in the reticulocyte lysate cell- free system. Second, since protein synthesis is programmed by endo- genous mRNA, it is not necessary to purify globin mRNA and add it to the incubation mixture. 'Finally, the lysate system does not require fractionation of the reticulocytes beyond lysis and removal of the cell membranes in its preparation. When compared to whole cell 176 incubation mixtures, the reticulocyte lysate is Vmore physiological" than more highly fractionated cell-free systems. After characterization of the reticulocyte lysate incubation mixture and determination of the incubation conditions which would yield maximum amounts of protein synthesis, a series of experiments was designed to determine if a limiting component of this system could be demonstrated as the origin of nascent globin peptide size accumu- lations. Preliminary experiments had determined that the nascent chain population from the lysate also demonstrated a nonuniform size distri- bution upon Bio-Gel A 0.5 m chromatographic analysis under denaturing conditions. Accumulations have been observed in nascent chain p0pula- tions purified from rabbit reticulocyte incubation mixtures of the same sizes as described for the whole cell incubations of Protzel and Morris (1974). The first component of the reticulocyte lysate cell-free protein synthesizing system to be investigated was tRNA. Transfer RNA was chosen initially because the interaction between tRNA isoacceptor Species and mRNA occurs at specific regions of the mRNA determined by the codons along the mRNA nucleotide sequence. It is possible to envision a tRNA species being present at such a low concentration that the ribosome involved in translation of the mRNA would have to pause when the ribosome reached the codon which interacted with that scarce tRNA. A slow step, if it existed, could cause an accumulation of nascent chains of the size corresponding to the codon directly prior to that involved with the tRNA in a low concentration. Reticulocyte tRNA was prepared by the method of Holley gt_gl, (1961) from the frozen reticulocyte lysate as described in Methods. 177 Lysate incubation mixtures were prepared which had received the addition of the purified tRNA. As described in the Results section, raising the tRNA concentration of the reticulocyte lysate incubation mixture to as much as five times the original concentration did not alter the elution profile obtained upon chromatographic analysis of the size distribution of the nascent chain population under denaturing condi- tions. The identical result was obtained in experiments utilizing different lysate incubation mixtures and different tRNA preparations. No alterations in the nascent chain accumulation pattern were observed. An eXperiment was also performed to examine the possibility that there did exist a limiting tRNA in reticulocytes at such low concen- trations that even an increase to five times its original concentration would not be sufficient to reduce the size accumulations observed in the Bio-Gel elution profile of the rabbit a— and B-globin nascent chain population. Transfer RNA was purified from rabbit liver by the method of Bose g__§l, (1971) and added to the reticulocyte lysate cell- free protein synthesis system. Nascent chains were radioactively labeled, purified and analyzed by Bio-Gel A 0.5 m chromatography. Addition of rabbit liver tRNA to the lysate incubation mixture gave the same result as the addition of reticulocyte tRNA. The gel elution profile was not altered. The size accumulations were still present in the radioactively labeled nascent chain p0pulation. Experiments involving tRNA from two different sources, the rabbit reticulocyte and rabbit liver, have therefore failed to alter the nascent globin chain size accumulations in the reticulocyte lysate incubation mixtures when the tRNA concentrations were brought to as much as five times the original level. The conclusion drawn from the 178 experiments involving the addition of tRNA preparations to the incuba- tion mixtures is that the nascent globin chain size accumulations are not due to a limitation of one or more tRNA isoacceptor species in the rabbit reticulocyte. After limiting amounts of tRNA had been eliminated as the possible origin of the globin nascent chain accumulations, an attempt was made to determine if other components of the reticulocyte lysate incubation were responsible for the size accumulations. Experiments were per- formed in an attempt to extract such a component from the reticulocyte lysate. The concentration of this component could then be increased to excess to a reticulocyte lysate incubation mixture. TWo procedures were used in the attempt to purify such a component. The first involved the preparation of a 0.5 M_KCl wash of ribosomes isolated from the reticulocyte lysate by centrifugation. The 0.5 M KCl salt wash preparation was added to a lysate incubation mixture in an analogous fashion to the experiments involving reticulocyte tRNA. Adjusting the concentration of the reticulocyte lysate incubation mixture to three times the endogenous level of these factors failed to alter the nascent chain accumulation pattern. The salt wash prepara- tion (a crude fraction containing many different proteins eluted from the ribosomes) did not contain a factor which was capable of altering the presence of nascent globin peptide size accumulations in the rabbit reticulocyte lysate incubation mixtures. A second experiment was performed in which the proteins precipi- tated from a 70% (NH4)2S04 reticulocyte lysate postribosomal super- natant were added to a lysate incubation mixture in excess of their normal concentration. ‘A lysate incubation mixture was adjusted to 179 three times the original concentration of the (NH4)2SO4 precipitated factors. Chromatographic analysis of the nascent chain population purified from this incubation showed, as described in Results, that no alteration of the Bio-Gel elution profile was observed. The material precipitated from a rabbit reticulocyte postribosomal supernatant by 70% (NH4)ZSO4 did not contain a component which was capable of altering the presence of the nascent chain size accumulations in the rabbit reticulocyte lysate cell-free synthesizing system. The attempts to identify a component of the reticulocyte lysate cell-free protein synthesizing incubation mixtures which is responsible for the nascent chain accumulations by being present in limiting amounts are all subject to the same reservation. That is, if such a factor does indeed exist, it may not have been purified during the procedures used in these eXperiments. Alternatively, the limiting factor could also have been inactivated by these purification proce- dures. If such a factor is either not purified or is inactivated, addition of the preparations of tRNA, ribosome salt wash factors, or postribosomal supernatant factors would not alter the concentration of the limiting component in the lysate incubation mixtures and would not be expected to affect the nascent chain size accumulations observed in the gel elution profiles. Due to the reservations described above with respect to the attempt to purify components of the reticulocyte lysate, an entirely separate and independent approach to the question has been devised. Instead of adding components of the lysate chain elongation system back to incubation mixtures, a method has been used which would increase the relative concentrations of all the chain elongation components relative to actively translating ribosomes. 180 The antibiotic, edeine, has been shown to specifically inhibit protein synthesis at the step of the initiation of mRNA translation (Obrig gt_al,, 1971). Inhibition of protein synthesis by edeine (as measured by a decrease in the rate of incorporation of a radioactive amino acid into TCA precipitable material) to 50% of its original rate would be expected to result in a 50% reduction in the number of active ribosomes involved in mRNA translation since edeine specifically inhibits the initiation and not elongation step of polypeptide synthe- sis. By the same reasoning, inhibition of the protein synthetic rate to 25% of the original value would result in a 75% reduction in the number of ribosomes actively translating the mRNA p0pulation. Reducing the amount of ribosomes involved in chain elongation by this method would have the effect of increasing the concentration of all the soluble components of the chain elongation system in relation to the active ribosomes. Although the absolute concentration of the components of peptide chain elongation in the lysate incubation mixtures would not be altered, the ratio between the elongation com- ponents and the ribosomes involved in chain elongation would be increased to four times its original value in incubations which have protein synthesis inhibited to 25% of its uninhibited rate by edeine. The experiment described above achieves the same effect as the experi- ments involving purification of lysate components and adding them back to lysate incubation mixtures. The concentration of a hypothetical factor of the chain elongation machinery which is postulated to be the origin of the nascent chain accumulations by being present in limiting amounts would be increased four times relative to the active ribosome concentration. The increase in relative concentration is accomplished 181 without purifying any components from the reticulocyte lysate. The possibility that a limiting component of the translation machinery has not been purified or that it has been inactivated is therefore eliminated. Edeine was added to a reticuloycte lysate incubation mixture at a concentration which had been determined to reduce the protein biosyn- thetic rate to 25% of its initial uninhibited value. Nascent globin chains were labeled and purified as described in Methods. The results of Bio-Gel A 0.5 m chromatographic analysis under denaturing conditions of the nascent a- and B-globin peptides indicated that there was no effect upon the nascent chain size accumulations by inhibiting protein biosynthesis to 25% of its original value by edeine. Two entirely separate approaches have, therefore, failed to demon- strate that the stoichiometrically limiting quantity of any component of the reticulocyte lysate cell-free protein synthesis system is the origin of the nascent chain size accumulations observed upon Bio-Gel chromatographic analysis of purified nascent chains under denaturing conditions. The conclusion has been reached that nascent chain size accumulations are associated with some other aspect of protein bio- synthesis than limiting components of the protein biosynthetic machinery. Since the origin of the nascent chain size accumulations did not seem to lie in the concentrations of the components of the protein biosynthetic machinery in the reticulocyte lysate, it was decided to determine if the presence of size accumulations in the nascent chain population was associated with the reticulocyte lysate incubation mixture itself, or was present in globin nascent chains isolated from another system in which globin mRNA was serving as the template for 182 protein biosynthesis. The effects of the translation system (the reticulocyte lysate) could, therefore, be separated from the effects of the template (the globin mRNA). The protein biosynthetic system chosen was the wheat embryo-derived cell-free protein biosynthetic system (Marcus gt_al,, 1975). The wheat embryo cell-free system had been shown previously to synthesize rabbit a- and B-globin from purified mRNA and had a low endogenous incorpora- tion rate (Efron and Marcus, 1973). Furthermore, the relatively large phylogenetic distance between rabbits and wheat helps to reduce the possibility that a unique property of the reticulocyte lysate protein biosynthetic machinery would also be present in the wheat embryo cell- free system with respect to the origin of the observed nascent chain accumulations. A prokaryotic system was not chosen because eukaryotic mRNA will only be translated at a low rate (if at all) in prokaryotic cell-free systems (Davies and Kaesberg, 1973). Globin mRNA was purified from rabbit reticulocytes as described in Methods using the oligo(dT)-cellulose chromatography procedure of Aviv and Leder (1972), and added to the wheat embryo-derived cell-free system as described in Results. Nascent chains were purified as described in Methods and analyzed by Bio-Gel A 0.5 m chromatography. The elution profile of nascent chains isolated from rabbit globin mRNA programmed protein biosynthesis in the wheat embryo-derived cell- free system is nearly identical to the profile obtained by chromato- graphic analysis of nascent chains isolated from the reticulocyte lysate cell-free system. The globin mRNA purification procedure involves the treatment of polysomes with 1% SDS and phenol. Proteins bound to the nucleic acids 183 will be denatured and removed by this treatment. The oligo(dT)- cellulose chromatography will separate the globin mRNA from other types of nucleic acids since rRNA and tRNA do not contain poly(A) regions in their primary sequence and will not bind to oligo(dT)-cellulose. Only rabbit globin mRNA is the common factor present in both the reticulo- cyte lysate and wheat embryo cell-free incubation mixtures. (This statement, of course, regards reticulocyte and wheat embryo ribosomes, elongation factors, tRNA, etc. as being "different".) The conclusion drawn from this experiment is that the origin of the nascent a- and B- globin chain size accumulations lies in the globin mRNA itself and not in the protein biosynthetic system in which it is being translated. Three separate approaches have been taken in the experiments described above: 1) it has not been possible to isolate a fraction from the rabbit reticulocyte lysate which is capable of affecting the nascent chain size distribution when added back to a lysate incubation mixture; 2) inhibition of the rate of initiation of protein biosyn- thesis in the reticulocyte lysate to 25% of its uninhibited rate (which will increase the concentration of protein synthesis elongation components by four times in relation to the number of actively trans- lating ribosomes) does not cause an alteration in the elution profiles observed upon gel chromatographic analysis of nascent chains purified from the partially inhibited reticulocyte lysate incubation mixtures; 3) accumulations of identical sizes are observed in the nascent chain populations isolated from the globin mRNA-directed wheat embryo- derived cell-free protein synthesis system in which the rabbit globin mRNA is the only component present that has been purified from the rabbit reticulocyte. The three types of experiments all lead to one 184 conclusion concerning the origin of the globin nascent chain size distributions: the origin of the nascent chain size accumulations must lie in some aspect of the globin mRNA structure and not in the translation system being used or the availability of the components needed for peptide chain elongation. As discussed in the Introduction, all RNA species which have been studied in both eukaryotes and prokaryotes contain regions of intra- molecular base pairing. Regions of secondary structure have been deduced to exist from physical, enzymatic, and sequence studies. Since globin mRNA had been shown to contain such regions by a variety of physical and enzymatic methods, it was felt that the origin of the size distributions observed in the chromatographic analysis of the rabbit a- and B-globin nascent chain p0pulation might be related to the presence of such structures. Such regions of secondary structure must be removed before a ribosome involved in translation of a mRNA could traverse a region involved in the base pairs. A ribosome might be expected to slow its rate of movement when the ribosome reached a base paired region during protein biosynthesis. The slowed movement would be the result of the ribosome either pushing through the double stranded region or waiting until the region became single stranded through a normal "breathing" of the molecule. In order to examine the hypothesis that mRNA secondary structure was the origin of the nascent chain size accumulations, it was neces- sary to be able to purify nascent peptide chains from the translation of a single mRNA. The nucleotide sequence of the mRNA being trans- lated must have been determined and a structure for this RNA sequence proposed. Chromatographic analysis of the nascent chains upon a 185 Bio-Gel A 0.5 m column under denaturing conditions would enable a comparison of the peptide lengths of any nascent chain size accumu- lations observed in the elution profile and the proposed structure. If the hypothesis that the nascent globin peptide size accumulations are associated with regions of secondary structure is correct, the regions of RNA secondary structure should be associated with the positions on the mRNA at which the rate of ribosome movement is relatively slow (as analyzed by the accumulation of nascent chains of discrete sizes). Only one mRNA met the requirements outlined above at the time the experiments described in this thesis were performed. The mRNA meeting the above requirements was the mRNA for bacteriophage M52 coat protein, which is also the M52 bacteriophage genome. It was possible, as shown in Resu1ts, to obtain 90% M52 coat protein biosyn- thesis after infection of E, ggli_with M82. The primary structure of the MS2 coat protein cistron (and the entire MS2 genome) had been determined and a structure proposed for the M52 coat protein RNA nucleotide sequence involving extensive regions of intramolecular base pairs (Min Jou 33 31,, 1972; Vandenberghe gE_al,, 1975; Fiers gt_gl,, 1975, 1976). The proposed structure of the M52 coat protein RNA (shown in Figure 1) involved the formation of a number of hairpin turns which could be expected to reform after translation of that region by a ribosome. Therefore, it was decided to detenmine the Bio-Gel elution profile for MS2 coat protein nascent chains purified from MSZ infected E, gglj_which were synthesizing essentially only MS2 coat protein. BacteriOphage M52 coat protein, 129 amino acids long, was of a small enough size that analysis of the nascent chain 186 elution profile could be performed upon the same Bio-Gel A 0.5 m chromatography column used for nascent globin peptide size distribu- tion analysis. Previous reports have demonstrated that treatment of bacteriophage M52 or f2 infected E, ggli_with either actinomycin D or rifampicin would result in cessation of host protein synthesis, but not affect bacteriOphage RNA coded protein synthesis (Vinuela eE_al,, 1967; Fromageot and Zinder, 1968). The bacteriophage RNA synthesis is not affected by rifampicin because its replication is catalyzed by a rifampicin-resistant RNA-dependent RNA polymerase distinct from the DNA dependent RNA polymerase used for E, ggli_RNA synthesis (Hartmann _e_t_a_l,, 1967). Using incubation conditions similar to those described by Froma- geot and Zinder (1968), the synthesis of bacteriophage M32 proteins was studied as described in Results. Protein synthesis in rifampicin treated E, gglj_was found to be increased to approximately twelve times the uninfected rate. Protein synthesis in rifampicin treated E, ggli_was dependent upon infection by bacteriOphage MS2. The rate of incorporation of 3H-leucine in M52 infected, rifampicin treated E, gglj_was linear from 20 to 65 minutes post-infection. M52 infected, rifampicin treated E, ggli_were labeled with 3H- 1eucine for a 15 minutes period starting 45 minutes after infection. The protein was isolated from the cells and analyzed by SOS-polyacryl- amide gel electrOphoresis. The products of protein biosynthesis were seen to be greater than 80% M52 coat protein. Therefore, the nascent chain population present in M52 infected, rifampicin treated E, coli 187 at 45 to 60 minutes after infection contained greater than 90% MS2 coat protein nascent chains. Purification of nascent MS2 coat protein chains from infected E, ggli_was described in Methods. Analysis of the size distribution of the nascent chain p0pulation by Bio-Gel A 0.5 m chromatography under denaturing conditions demonstrated a nonuniform gel elution profile. Nascent chain accumulations of identical sizes were evident in profiles 3H-leucine and 3H-aspartic acid, of nascent chains labeled with both indicating that the size accumulations are independent of the radio- active label used, as is the case for nascent a- and B-globin nascent chains described earlier in this thesis. Translation of different mRNA yielded unique patterns of nascent chain size accumulations, as would be predicted if the origins of those accumulations resided in the mRNA itself. In contrast to the observations of the elution profile obtained by chromatographic analysis of nascent chains purified from M52 infected, rifampicin treated E, 9911, the elution pattern of nascent chains isolated from an E, ggli_culture which had not been treated with either bacteriophage MS2 or rifampicin showed very few regions of nascent chain accumulations. There were no size accumulations in the region of M52 nascent chain peptides (Kd greater than 0.25). Those accumulations, which were present in the elution profile of nascent chains purified from uninfected E, gglj,were thought to be due to either E, cgli_proteins made in relatively large amounts or to ribo- somal proteins which adsorb to the DEAE-cellulose column under the isolation conditions. Because synthesis of E, coli proteins was inhibited by rifampicin addition, host nascent chains could not have 188 contributed to the MS2 coat protein nascent chain elution profile. Since a population of nascent chains obtained from a protein syn- thesizing sys?em is being analyzed, the lengths of the M52 coat protein nascent chains which are found to accumulate in the p0pulation of nascent M52 peptides may be estimated from their molecular weight ranges (Table 12) using the amino acid sequence of the MS2 coat protein (Dayhoff, 1972). The population of nascent chains of different sizes will all start at the N-terminal end of the coat protein amino acid sequence. The molecular weights of the possible 129 peptides which could be present in a MS2 coat protein nascent chain population have been determined by summing the molecular weights of the peptide residues starting with the N-terminal alanine. In such calculations the presence of an N-terminal formylmethionine in some of the peptides has not been included, although its presence is noted. The result of converting the molecular weight ranges of Table 11 to amino acid lengths is shown in Table 12. As described previously, one consequence of the relationship between the Kd and the molecular weight of the peptide in the elution profile is an increasing s10pe of the elution profile as the Kd value decreases (Protzel and Morris, 1974). The increasing slope of the elution profile arises since more members of the nascent peptide popu- lation are being collected per unit of volume at low Kd than at high Kd‘ A maximum in the elution profile will occur near the Kd associated with the molecular weight of the completed protein (Kd = 0.25, i.e., Peak VII in the case of coat protein. Still another factor which affects the general elution pattern of labeled nascent peptides is the location of the particular amino 189 Table 12. Amino acid lengths of M52 coat protein nascent chain accumulations calculated from the data in Table 11. a . c Length of. d Peak Number Kd + 5.0. Molecular Ne1ght Nascent Pept1de I 0.80 s 0.01b 1,280-1 .444 11-14 II 0.72 i 0.01 2,005-2,223 20-22 III 0.63 i 0.005 3,155-3,296 31-32 IV 0.52 i 0.01 4,869-5,261 47-50 V 0.39 i 0.01 7,907-8,495 75-81 VI 0.33 i 0.005 9,973-10,337 93-96 VII 0.25 i 0.005 13,293-13,789 125-130 aData presented here summarize the results of nine analyses of the size distribution of nascent coat protein peptides. The distri- bution coefficient ( ) of each maximum in the Bio-Gel A 0.5 m elution profile is in icated (see, for example, Figure 5). bDetected as a minor peak in five of the nine experiments. cThe range of molecular weights indicated by the standard deviation associated with each K value was calculated using the values ob— tained from a Bio-Gel 0.5 m column standardized with M52 coat protein and the peptides derived from MS2 coat protein by cyanogen bromide cleavage (12). dThe length of nascent peptides corresponding to the calculated molecular weights was determined using the amino acid sequence of M52 coat protein and the molecular weight of each amino acid resi- due involved, beginning with the N-terminal residue of the mature coat protein. An average molecular weight of 110 was used for residue 130 of peak VII. 190 acid used as the source of radioactivity in the amino acid sequence of the nascent peptide. The grouping of seven leucine residues in the region of peptide lengths corresponding to Kd values of 0.391 to 0.282 undoubtedly contributed to the amplitude of the peak seen at Kd = 0.25 in Figure 31. Nascent M52 peptides of approximately 11-14, 20-22, 31-32, 47-50, 75-81, and 91-96 amino acid residues in length accumulate in the popu- lation of nascent M32 peptides. The large peak of labeled nascent peptides seen at Kd = 0.25 is thought to be a product of the properties of the Bio-Gel column and the particular labeled amino acid used as described above. From the pr0posed "Flower Model" for the M32 coat protein gene, one may determine the codons which direct the synthesis of peptides of the lengths indicated in Table 12 and also compare those locations along the base sequence of the M32 coat protein genome with the pr0posed regions of secondary structure in the M32 RNA. These data are summarized pictorially in Figure 44. A consideration of the relative stability of the proposed regions of secondary structure of the ”Flower Model" indicates that the two smaller "hairpin" 100ps between regions VI and VII are probably not stable at the temperature at which the incubations were conducted (Min Jou gt_al,, 1972). However, the small hairpin loop immediately following region IV of Figure 44 would be expected to be stable if the structure were written in a slightly different manner. The stable arrangement would place four base pairs in the stem and six nucleo- tides in an unbounded loop. This arrangement has a calculated free energy value of -6.4 kcal per mole at 25° using the stability estimates 191 .mucmscmm wuwuom.uzc cocumwu cwmpoca pmou mm: ms“ co ucogmmceou mcowum.:E:uum cwmcu ucmummc sows: cu mcowpmmoa mzh .ee ee=m_a 193 ser molecule of Tinoco gt_gl, (1971, 1973). A structure in a yeast tRNA containing a similar stem of four base pairs has been found to have a Tm of 83° (Coutts, 1971). An alternate structure for the region between I and II has been proposed which would place an additional "hairpin" loop of moderate stability immediately following region I of Figure 44 (Min Jou gE_al,, 1972). This alternate structure is shown in Figure 45. Correlation of the size of nascent peptides which accumulate during M32 coat protein biosynthesis and the position of the ribosome carrying those lengths of nascent peptides in the flower model of the M32 genome reveals that five of the six regions along the M32 coat protein RNA where accumulations of nascent peptides occur during translation (i.e., I, II, III, IV, and V) occur at, or very near, a point where the ribosome involved in translation of the RNA would be entering a predicted region of RNA secondary structure in the M32 genome. Region VI of Figure 44 does not correlate with the beginning of a region of double stranded structure of M32 RNA according to the model as shown. Region VI, however, is immediately 5' to a region of the M32 structure for which alternative structures may exist, a region where secondary structures has been considered as "highly tentative" by the authors of the "Flower Model". This highly tentative region includes the potentially stable structure immediately preceding region VII. Since the "Flower Model" for the coat protein of M32 has been predicted on theoretical grounds using the base sequence of only that region of M32 and not the complete sequence of M32, which is now known to contain some 3,569 nucleotide residues, the proposed secondary structure for the coat protein region may be oversimplified. 194 .cocpm_u cwmpocq pmou mm: 82p mo cowpcoa m so» wczuuscpm mpmccmppm c< .me 6555.. 195 R 000 0044000000 <40. 000‘ £033000 < 00440404104440-1119 5 e s 6 :81 . 196 Consequently, secondary structural effects of the other regions of M32 RNA upon the rate of translation of the coat protein gene have not been considered here. Neither have attempts been made to take into account possible ribosome-ribosome interactions, or queuing, upon a given mRNA during the translation of the coat protein region since such interaction might be expected to hold open regions of an mRNA which might otherwise return to a double structure between each round of translation (von Heijne gal. , 1977). Lodish and Robertson (1969) have provided evidence that ini- tiation of translation of the RNA polymerase gene of M32 RNA may occur only when the initiation of codon for that gene is liberated from a region of secondary structure of M32 RNA by translation of the coat protein gene (See Figure l and 45). The data presented in this thesis suggest that the effects of secondary structure are more general and have the ability to alter the rates of chain elongation as well by altering the rates of translation of specific regions of the mRNA. The report that the in_!jtrg rates of bacteriophage f2 coat protein synthesis, when measured between the 3rd and the 70th codons and the 3rd and 129 codons, are identical is not incompatible with the data reported here (Webster and Zinder, 1969). The observations presented in the present experiments involve the measurement of rela- tive rates of nascent peptide chain elongation between different codons along the mRNA. The rates of chain elongation measured by Webster and Zinder are averages of the chain elongation rate for a number of successive elongation steps and may well include a relative nonuniformity of elongation rates within the regions studied. 197 Measurement of the jn_glxg_nascent chain elongation rate of E, ggli_ protein synthesis yields a value of about 1000 amino acids per minute at 37° (Forchhammer and Lindahl, 1971). Eg_gitgg_studies have shown that the rate of T4 lysozyme synthesis is 180 amino acids per second at 31° (Wilhelm and Haselkorn, 1970), and the rate of f2 coat protein chain elongation at 33° is 30 amino acids per minute (Webster and Zinder, 1969). Thus, previously measured rates of chain elongation are seen to vary for different mRNA's with the RNA bacteriophage coat protein elongation rate being much lower than that of other proteins. The suggestion has been made that this lower elongation rate during f2 coat protein synthesis is due.to the phase RNA secondary structure (Wilhelm and Haselkorn, 1970; Capecchi and Webster, 1975). However, the means by which this might occur was not clear. The first indi- cation of the validity of this idea has been presented in this thesis. The data presented in this thesis have led to the bypothsis that the origin of nascent chain size accumulations during chain elongation arises as a result of the slowing of the rate of ribosome movement along the mRNA by mRNA secondary structure. As ribosomes reside in certain regions (those preceding the areas of mRNA secondary structure) longer than other regions, the nascent chain population purified from these ribosomes will show size accumulations upon a chromatographic analysis by elution from Bio-Gel A 0.5 m under denaturing conditions. Although the correlation between bacteriOphage M32 coat protein nascent chain accumulations and the proposed secondary structure of the M32 coat protein cistron is quite good (five of six points of accumulation correspond to the beginning of a region of a potentially stable region of secondary structure), the extent to which this 198 hypothesis is applicable to other mRNA Species must be determined. Since the nascent chain accumulations have been shown in this thesis to be associated with the mRNA itself, and not the translation system (for globin mRNA), it would be possible to determine the Bio-Gel elution pattern for any mRNA which has been purified by translation of that mRNA in the wheat germ cell-free system. Alternatively, the mRNA-dependent reticulocyte lysate cell-free system prepared by nuclease treatment could be used (Pelham and Jackson, 1976). Such data, coupled with the nucleotide sequences of the mRNA, could allow compari- son of the potential regions of secondary structure of the mRNA with the regions at which ribosomes demonstrate slower rates of chain elongation as measured by nascent chain accumulations. The existence of more examples of such correlations of nascent chain Size accumu- lations with regions of potential mRNA secondary structure would give further support to this hypothesis. Direct analysis of the regions of mRNA which are involved in secondary structure is another useful approach. If regions of secondary structure can be shown to exist in defined areas of the mRNA by physical or enzymatic means, the correlation with the nascent chain Size accumu- lations in a manner similar to that described in this thesis for M32 coat protein would provide independent evidence in support of the hypothesis expressed here. Such analyses are now being undertaken for rabbit a- and B-globin mRNA in the laboratory in which the work reported in this thesis was performed. A third approach to the question of the effects of mRNA secondary structure upon chain elongation would be to perform jn_xlt§g_experi- ments using a bacterial mRNA-dependent cell-free protein synthesis 199 system and bacteriOphage RNA. In an analogous manner to the rabbit a- and B-globin Size accumulation analysis, the M32 coat protein nascent chair elution profile was first investigated jn_gixg, Analysis in a cell-free system would allow the closer study of the size accumu- lations in the coat protein nascent chain p0pu1ation. The hypothesis proposed as a result of the experiments conducted in this thesis, that the origin of the nascent chain size accumula- tions observed upon Bio-Gel chromatographic analysis under denaturing conditions of a nascent chain p0pulation lies in the mRNA secondary structure, is the simplest explanation of the data presented. Other interactions such as limiting tRNA concentrations, mRNA binding proteins, effects of tRNA isoacceptor Species upon the rate of the peptidyl transferase reaction, obstruction of the ribosome by a complex folding of the mRNA, etc., could be postulated to have Similar effects in the translation of an mRNA. 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