Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes.

We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast. The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes. We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14%. The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme. Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain. The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective. The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe). The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate. The fact that the enzyme hydrolyzes E. coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).     

The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3'-AMP terminus of susceptible tRNA substrates.

The preceding paper (Gross, M., Starn, T.K., Rundquist, C., Crow, P., White, J., Olin, A., and Wagner, T. (1992) J. Biol. Chem. 267, 2073-2079) reported the purification and partial characterization of rabbit reticulocyte peptidyl-tRNA hydrolase. In this article we demonstrate that, unlike bacterial and yeast peptidyl-tRNA hydrolase which act by deacylation, the reticulocyte enzyme hydrolyzes N-acylaminoacyl-tRNA to N-acylaminoacyl-AMP. Reticulocyte lysate has a separate enzyme, that we have isolated and termed aminoacyl-AMP deacylase, which hydrolyzes N-acylaminoacyl-AMP and aminoacyl-AMP, recycling the amino acid and nucleotide components. The action of this enzyme is relatively specific for the N-acylaminoacyl-AMP generated by peptidyl-tRNA hydrolase, since it is much less active with N-acylaminoacyl-adenosine and inactive with N-acylaminoacyl-ACCAC, N-acylaminoacyl-tRNA, or aminoacyl-tRNA. The tRNA product of peptidyl-tRNA hydrolase action is tRNA missing only its 3'-AMP terminus (tRNA(c-c)), since reaminoacylation requires tRNA nucleotidyltransferase but not CTP. The 3' exonucleolytic action of reticulocyte peptidyl-tRNA hydrolase is specific to susceptible tRNA substrates, since it does not hydrolyze CACCA, CACCA-N-acylamino acid, polyuridylic acid, or the 3' polyadenylate tail of globin mRNA, and, since its ability to hydrolyze Escherichia coli f[3H]Met-tRNA(fMet) is not reduced by excess 5 S or 28 S ribosomal RNA and is reduced only slightly by excess tRNA(c-c). Reticulocyte peptidyl-tRNA hydrolase also hydrolyzes th 3'-AMP terminus of deacylated tRNA. This property may explain why the 3'-terminal AMP of tRNA undergoes turnover in reticulocytes and reticulocyte lysate, since we find that such turnover in gel-filtered reticulocyte lysate is increased under conditions where aminoacylation is reduced.     

Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.     

The efficiency of methionine incorporation from isoaccepting species of tRNAMet into rabbit globin in an homologous reticulocyte lysate system.

Rabbit globin alpha and beta chains were labeled with [3H]leucine, and with [35S] -methionine from reticulocyte tRNAMet isoacceptors using a rabbit reticulocyte cell-free synthesis system. [35S]Methionine from the three tRNAMet species isolated by RPC-5 chromatography was incorporated into internal positions of both alpha and beta globin. The initiator tRNA, tRNAIMet, exhibited very low efficiency for incorporating methionine internally, while tRNAIIMet was four times more efficient than tRNAIIIMet. Amino acid analysis of the tryptic peptides of the labeled globins revealed that all three isoacceptors incorporated methionine into the normal methionine peptides. Similar studies with Escherichia coli [35S]Met-tRNAfMet showed a 3-fold increase over the reticulocyte initiator tRNA in its capacity to incorporate methionine into the internal positions of rabbit globin.     

The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.     

Function of the extra 5'-phosphate carried by histidine tRNA

Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Receptor site for the 5'-phosphate of elongator tRNAs governs substrate selection by peptidyl-tRNA hydrolase

Eubacterial peptidyl-tRNA hydrolase (PTH) recycles all N-blocked aminoacyl-tRNA molecules but initiator formyl-methionyl-tRNAfMet, the acceptor helix of which is characterized by a 1-72 mismatch. Positive selection by PTH of noninitiator tRNA molecules with a full 1-72 base pair is abolished, however, upon the removal of the 5'-phosphate. The tRNA 5'-phosphate plays therefore the role of a relay between the enzyme and the status of the 1-72 base pair. In this study, the receptor site for the 5'-phosphate of elongator peptidyl-tRNAs and the position at the surface of PTH of the 3'-end of complexed peptidyl-tRNA are identified by site-directed mutagenesis experiments. The former site comprehends two cationic side chains (K105 and R133) which are likely to clamp the phosphate. The second corresponds to a four asparagine cluster (N10, N21, N68, and N114). By using these two positional constraints, the acceptor arm of elongation factor Tu-bound Phe-tRNAPhe could be docked to PTH. Contacts involve the acceptor and TPsiC stems. By comparing the obtained 3D model to that of EF-Tu:Phe-tRNAPhe crystalline complex in which the 5'-phosphate of the ligand also lies between a K and an R side chain, we propose that, in both systems, the capacity of the 5'-phosphate of a tRNA to reach or not a receptor site is the main identity element governing generic selection of elongator tRNAs. On the other hand, while the 1-72 mismatch acts as an antideterminant for PTH or EF-Tu recognition, it behaves as a positive determinant for the formylation of initiator Met-tRNAfMet.     

Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation

The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.     

Recognition of the initiator tRNA by the Pseudomonas aeruginosa methionyl-tRNA formyltransferase: importance of the base-base mismatch at the end of the acceptor stem

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) in eubacteria is catalyzed by methionyl-tRNA formyltransferase (MTF). Features of the Escherichia coli tRNAfMet that are important for formylation are the base-base mismatch between nucleotides 1 and 72, and the second and third base pairs of the acceptor stem. The base-base mismatch is the most crucial formylation determinant in the E. coli tRNAfMet. However, it is not known whether this feature is also important for formylation of other eubacterial tRNAfMet. We cloned the Pseudomonas aeruginosa MTF gene by complementation of an E. coli MTF mutant strain with a genomic library, and investigated the catalytic properties and substrate specificity of the enzyme. The results show that the P. aeruginosa and E. coli enzymes have comparable affinities for the tRNAfMet and N10-formyltetrahydrofolate (fTHF) substrates. Overproduction of the P. aeruginosa MTF rescued the initiator activity of an E. coli formylation-defective tRNAfMet with a base pair between nucleotides 1 and 72, indicating that the base-base mismatch is utilized by the P. aeruginosa MTF for recognition of the tRNAfMet. Therefore, this feature may be used by MTFs from other eubacteria to distinguish the initiator from elongator tRNAs.     

Mammalian mitochondrial initiation factor 2 supports yeast mitochondrial translation without formylated initiator tRNA

Initiation of protein synthesis in mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl-tRNA (fMet-tRNAfMet) in a process involving initiation factor 2 (IF2). However, yeast strains disrupted at the FMT1 locus, encoding mitochondrial methionyl-tRNA formyltransferase, lack detectable fMet-tRNAfMet but exhibit normal mitochondrial function as evidenced by normal growth on non-fermentable carbon sources. Here we show that mitochondrial translation products in Saccharomyces cerevisiae were synthesized in the absence of formylated initiator tRNA. ifm1 mutants, lacking the mitochondrial initiation factor 2 (mIF2), are unable to respire, indicative of defective mitochondrial protein synthesis, but their respiratory defect could be complemented by plasmid-borne copies of either the yeast IFM1 gene or a cDNA encoding bovine mIF2. Moreover, the bovine mIF2 sustained normal respiration in ifm1 fmt1 double mutants. Bovine mIF2 supported the same pattern of mitochondrial translation products as yeast mIF2, and the pattern did not change in cells lacking formylated Met-tRNAfMet. Mutant yeast lacking any mIF2 retained the ability to synthesize low levels of a subset of mitochondrially encoded proteins. The ifm1 null mutant was used to analyze the domain structure of yeast mIF2. Contrary to a previous report, the C terminus of yeast mIF2 is required for its function in vivo, whereas the N-terminal domain could be deleted. Our results indicate that formylation of initiator methionyl-tRNA is not required for mitochondrial protein synthesis. The ability of bovine mIF2 to support mitochondrial translation in the yeast fmt1 mutant suggests that this phenomenon may extend to mammalian mitochondria as well.     

How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis

During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNAfMet bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.     

The role of modified purine 64 in initiator/elongator discrimination of tRNA(iMet) from yeast and wheat germ.

The role of 2'-ribosylated adenosine 64 in tRNA(iMet) from yeast in initiation/elongation discrimination was investigated. As measured by in vitro translation in rabbit reticulocyte lysate, the specific removal of the 2'-ribosylphosphate at adenosine 64 via periodate oxidation allows tRNA(iMet) to read internal AUG codons of the globine messenger RNA. Yeast Met-tRNA(iMet) lacking the modification of nucleoside 64 forms ternary complexes with GTP and elongation factor Tu from Escherichia coli. The lack of modification at position 64 does not prevent tRNA(iMet) from participating in the initiation process of in vitro protein synthesis. Wheat germ tRNA(iMet) has a 2'-ribosylated guanosine at position 64. Removal of this modification from the wheat germ tRNA(iMet) enables it to read internal AUG codons of globine and tobacco mosaic virus messenger RNA in reticulocyte and wheat germ translation systems, respectively.     

Affinity labeling of eukaryotic elongation factors using N epsilon-bromoacetyl-Lys-tRNA.

eEF-T and eEF-Tu from rabbit reticulocyte and from Artemia were affinity labeled using N epsilon-bromoacetyl-Lys-tRNA prepared with either yeast or E. coli tRNA. Only the eEF-Tu polypeptide was crosslinked when eEF-T was incubated with the reactive aminoacyl-tRNA analogue, which indicates that at least part of the aminoacyl-tRNA binding site is the same in both eEF-Tu and the multisubunit eEF-T. Complex formation (eEF-Tu x aa-tRNA x GTP) was required for crosslinking, since no covalent reaction with eEF-Tu occurred in the absence of GTP. The yield of crosslinked product was greatly reduced by adding either unmodified rabbit liver aminoacyl-tRNA or unmodified E. coli Lys-tRNA to the incubation to compete for the aminoacyl-tRNA binding site on eEF-T or eEF-Tu, indicating that the covalent reaction occurs while the N epsilon-bromoacetyl-Lys-tRNA is bound in this site. The affinity labeling of a prokaryotic and two different eukaryotic elongation factors by the same reagent suggests that there may be conservation of structure in the region of the proteins which binds the aminoacyl end of the aminoacyl-tRNA.     

Mechanism of inhibition of eukaryotic translational initiation by the trinucleotide ApUpG

ApUpG, the oligoribonucleotide homologous to the initiation codon, as well as the tetranucleotides ApUpGpA and ApUpGpG block initiation of protein synthesis in the rabbit reticulocyte lysate. These oligonucleotides are recognized as translational initiation sites by the ribosomes, leading to a very large accumulation of complete, but inactive, 80 S initiation complexes, containing methionylated initiator tRNA and ApUpG in a 1:1 stoichiometry. ApUpG appears to inhibit by competing with endogenous globin mRNA for 80 S ribosomal couples, since the inhibition of protein synthesis by ApUpG can be largely relieved by increasing the globin mRNA. The 80 S · Met-tRNA_i^Met · ApUpG complexes are not formed in the absence of hemin, demonstrating that their formation requires the active recycling of eukaryotic initiation factor 2. In addition the trinucleotide correctly directs the Met-tRNA_i^Met into the ribosomal donor site, since the methionyl residue is puromycin-reactive.     

Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.     

Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages

The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNA(Phe) hydrolysis. The strong D-loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNA(Phe), while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNA(Phe). Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D-loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5' side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3' side of the loop. The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs.     

Primary and spatial structure of tRNA

The recent achievements in studying of structure of tRNA are considered in the present paper. A brief analysis of the new methods for sequencing tRNA was carried out. Due to the development of these methods about 300 tRNA primary structures have been determined. Comparison of the primary tRNA structures gives us the possibility to divide them into seven classes: prokaryotic initiator tRNAs and eukaryotic initiator tRNAs; prokaryotic elongator tRNAs and eukaryotic elongator tRNAs; archaebacterial tRNAs; and mitochondrial tRNAs of lower and higher eukaryotes. Structural properties of the tRNAs of each of these classes are discussed. The second part of the paper is devoted to the three-dimensional structure of tRNA. Recent data in this field obtained by X-ray crystallographic technique as well as by high-resolution NMR and chemical modification methods are reviewed.     

Assembly of M13 and M13am8H1R1 procoat protein into microsomes is stimulated by rabbit reticulocyte lysate and ATP

Processing of M13 procoat protein to transmembrane coat protein by dog pancreas microsomes is stimulated by a component of rabbit reticulocyte lysate and ATP. We asked whether this ATP-dependent reaction, involved in membrane assembly of procoat protein in the eukaryotic system, is related to the membrane potential dependent reaction observed for the membrane assembly of procoat protein in E. coli. Specifically, we asked if a mutant procoat protein which had been previously shown to be independent of the membrane potential with respect to its assembly in E. coli (M13am8H1R1 procoat protein) shows a stimulation by reticulocyte lysate and ATP in its assembly into microsomes. Since the mutant procoat protein behaved exactly as the wild type procoat protein in the eukaryotic in vitro system, we propose that the ATP-dependent reaction observed for the eukaryotic system does not substitute for the membrane potential dependent reaction in the prokaryotic system.     

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.