A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA _inff ^supMet . Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA _inff ^supMet . Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU₃ as mRNA, elongator-tRNAs competed with fMet-fRNA _inff ^supMet and with tRNA _inff ^supMet . fMet-tRNA _inff ^supMet reacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU₃ complex at the A site only if fMet-tRNA _inff ^supMet or tRNA _inff ^supMet filled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

On the accessibility and selection of the initiator site of mRNA in protein synthesis

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the “accessibility” of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

Purification and characterization of yeast mitochondrial initiation factor 2

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.     

Interaction between non-formylated initiator Met-tRNA(fMet) and the ribosomal A-site from Escherichia coli

We report studies in vitro of the interaction between non-formylated initiator Met-tRNA(fMet) and 70S ribosomes. The binding of Met-tRNA(fMet) to ribosomes carrying fMet-tRNA(fMet) in the P-site is strongly stimulated by elongation factor EF-Tu:GTP in the presence of (AUG)3. The enzymatically bound Met-tRNA(fMet) does not react with puromycin. The bound Met-tRNA(fMet) can accept formylmethionine from P-site-bound fMet-tRNA(fMet). These results demonstrate a functionally active binding at the ribosomal A-site. Partial ribonuclease digestion (footprinting) was used to study the sites in Met-tRNA(fMet) which are involved in the interaction with the ribosomal A-site. The results show that a large part of the tRNA molecule is protected by the ribosome against ribonuclease digestion. In addition to the protection found in the amino acid region and the anticodon arm, protection is seen in the D-loop and in the extra arm. No region within the bound tRNA is found to be more accessible for RNases than in the free Met-tRNA(fMet). The reported enhancement of ribonuclease cuts in the D- and T-arms of A-site-bound Phe-tRNAPhe is thus not found in A-site bound Met-tRNA(fMet).     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

An electron microscopic analysis of pathways for bacteriophage T4 DNA recombination

The interaction between ribosomes of Bacillus stearothermophilus and the RNA genomes of R17 and Qβ bacteriophage has been studied. Whereas Escherichia coli ribosomes can initiate the synthesis of all three RNA phage-specific proteins in vitro, ribosomes of B. stearothermophilus were previously shown to recognize only the A (or maturation) protein initiation site of f2 or R17 RNA. Under these same conditions, a Qβ region is bound and protected from nuclease digestion. Qβ RNA, however, does not direct the synthesis of any formylmethionyl dipeptide in the presence of B. stearothermophilus ribosomes, nor does the binding of either this Qβ region or the R17 A protein initiation site to these ribosomes show the same fMet-tRNA requirement for recognition of initiator regions as that previously established with E. coli ribosomes. Analysis of a 38-nucleotide sequence in the protected Qβ region reveals no AUG or GUG initiator codon. These observations suggest that messenger RNA may be recognized and bound by B. stearothermophilus ribosomes quite independently of polypeptide chain initiation.Binding experiments using R17 RNA and mixtures of components from B. stearothermophilus and E. coli ribosomes confirm the conclusion drawn by Lodish (1970a) that specificity in the selection of authentic phage initiator regions by the two species resides in the ribosomal subunit(s). However, anomalous attachment of B. stearothermophilus ribosomes to R17 RNA, which is observed upon lowering the incubation temperature of the binding reaction, is clearly a property of the initiation factor fraction. The results are discussed with respect to current ideas on the role of ribosomes and initiation factors in determining the specificity of polypeptide chain initiation.     

The effects of initiation factor 3 on the formation of 30S initiation complexes with synthetic and natural messengers

Initiation factor IF-3 is required for the binding of fMet-tRNA to 70S ribosomes directed by AUG, poly (U,G), f₂RNA and T₄ late RNA as well as for the binding of acPhe-tRNA directed by poly (U). In contrast, IF-3 is not required for the binding of the initiator aminoacyl-tRNAs to isolated 30S subunits directed by the synthetic messengers, but is required for maximal formation of initiation complexes with natural messengers. These data indicate that with synthetic messengers the sole function of IF-3 is to dissociate the 70S ribosomes into subunits, whereas with natural messengers IF-3 is required not only for dissociation of the ribosomes but also for the binding of the messenger to the 30S subunit.     

Structural features that underlie the use of bacterial Met-tRNAfMet primarily as an elongator in eukaryotic protein synthesis

Met-tRNAfMet from Escherichia coli is utilized efficiently as an elongator tRNA during protein synthesis in the rabbit reticulocyte lysate since it rapidly incorporates its methionyl residue into the same tryptic peptides of rabbit globin as the endogenous Met-tRNAmMet. Therefore, it must lack the structural characteristics that prevent the eukaryotic initiator tRNA from entering elongation. In contrast, E. coli Met-tRNAfMet appears to initiate very poorly since, unlike reticulocyte Met-tRNAiMet, it forms no detectable 43 S preinitiation complexes, and only a very small fraction of the methionine it contributes to polyribosomal peptidyl-tRNA is found at the N terminus. The bacterial fMet-tRNAfMet, which cannot elongate, is utilized for polypeptide chain initiation at a much lower level than the formylated Met-tRNAiMet from eukaryotes. The ability of E. coli Met-tRNAfMet to be used as an elongator and fMet-tRNAfMet as an initiator in the reticulocyte lysate may be considerably underestimated because of the rapid enzymatic hydrolysis of these initiator tRNAs in the lysate. The enzyme hydrolyzes fMet-tRNAfMet and Met-tRNAfMet from E. coli in a strictly Mg2+-dependent manner but not the corresponding species from yeast or rabbit reticulocytes. It also hydrolyzes yeast N-acetyl-Phe-tRNAPhe and reticulocyte peptidyl-tRNA, showing that this enzyme--like the eukaryotic protein synthetic machinery--does not readily distinguish the bacterial tRNAfMet from eukaryotic elongator tRNA.     

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5'-terminal AUG

Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage λ, with a 4-thiouridine (4^S-U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 5′-AUG is required for stable binding. Furthermore, the addition of a 5′-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.     

Escherichia coli ribosomes bind to non-initiator sites of Q beta RNA in the absence of formylmethionyl-tRNA.

Escherichia coli ribosomes and Qβ [³²P]RNA were incubated with or without fMet-tRNA under protein initiation conditions, treated with RNase A, and centrifuged through a sucrose density gradient. The sample incubated with fMet-tRNA gave a main radioactivity peak in the 70 S region, which consisted predominantly of coat cistron initiator fragments. After incubation without fMet-tRNA, equal amounts of radioactivity were found in the 70 S and the 30 S regions, but in both peaks almost all of the radioactivity was duo to three RNase A-resistant oligonucleotides, A-G-A-G-G-A-G-G-Up (P-2a), A-G-G-G-G-G-Up (P-15) and G-G-A-A-G-G-A-G-Cp (P-4). These three oligonucleotides are derived from three different RNA regions, none of which is close to a protein initiation site. All three fragments show striking complementarity to the 3′-terminal region of E. coli 16 S RNA. Ribosomes incubated with an RNase A digest of Qβ [³²P]RNA bound almost exclusively oligonucleotide P-2a; treatment with cloacin DF13 cleaved off a complex consisting of a 49-nucleotide long segment of 16 S rRNA and oligonucleotide P-2a. These experiments show that the interaction of 30 S ribosomes with the “Shine-Dalgarno” region preceding the initiator codon of the Qβ coat cistron is insufficient to direct correct placement of the ribosome on the viral RNA, and that an additional contribution from the interaction of fMet-tRNA with the initiator triplet is required for ribosome binding to the initiator region.     

Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.     

Features of 80S mammalian ribosome and its subunits

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (P_i), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (K_d 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic.     

Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast

Chemically formylated Met-tRNA_m^Met and Met-tRNA_f^Met species from $\text{E.}$$\text{coli}$ and yeast were tested for their capacity to serve as chain-initiators in a cell-free system from $\text{E.}$$\text{coli}$. In the presence of R 17 mRNA, initiation factors and $\text{E.}$$\text{coli}$ ribosomes, all four Met-tRNAs could form functional initiation complexes as measured by ribosomal binding kinetics, fMet-puromycin formation and synthesis of a dipeptide fMet-Ala. Unformylated Met-tRNA_f^Met from $\text{E.}$$\text{coli}$ displayed significantly less activity as a peptide chain-initiator than the formylated Met-tRNA_m^Met species from $\text{E.}$$\text{coli}$ and yeast. Although the latter tRNAs were less effective initiators than the “physiological” initiator tRNAs, the data seem to indicate that a blocked α-amino group represents the major token of identification by which Met-tRNA is admitted to function in $\text{E.}$$\text{coli}$ peptide chain initiation.     

Relationship between size of mRNA ribosomal binding site and initiation factor function

The rate and the extent of the binding of initiator fMet-tRNA(fMet) to 30S ribosomal subunits in the presence of IF1, IF2 and GTP is either inhibited or slightly stimulated by the presence of IF3 depending on whether the initiation triplet AUG or the polynucleotide poly(AUG) is used as template. To determine the length of the template required for the transition from the AUG- to the poly(AUG)-type of behavior in the presence of IF3, the ribosomal binding of fMet-tRNA was studied in response to AUG triplets extended on either the 5'- or the 3'-side by stretches of homo-oligonucleotides of different lengths. When the binding of fMet-tRNA was studied at equilibrium it was found that IF3 no longer inhibits the amount of ternary complex formed if AUG is extended either 10 nucleotides on the 5'- or 35-40 nucleotides on the 3'-side. When the initial rate of ternary complex formation is considered, shorter extensions (4 nucleotides on the 5'-side or 20-30 nucleotides on the 3'-side) are sufficient to elicit a substantial stimulation by IF3. These results are discussed in relation to the mechanism of action of the initiation factors in the selection of the initiation region of the mRNA by ribosomes.     

Identification and initial characterization of translational initiation factor 2 from bovine mitochondria

The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria. This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure. IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography. IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E. coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes. The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP. Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.     

On the accessibility and selection of the initiator site of mRNA in protein synthesis.

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the "accessibility" of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

Initiation factor 3 requirement for the formation of initiation complexes with synthetic oligonucleotides

Initiation factor IF-3 is required for the poly (U)-directed binding of N-acetyl-Phe-tRNA to 70S ribosomes as well as for the binding of fMet-tRNA directed by poly (U,G), AUG, and bacteriophage f₂ RNA. The formation of the 70S initiation complex is dependent upon IF-2 and is stimulated by IF-1. The requirement for IF-3 is not alleviated by high concentrations of the synthetic templates.     

Vaccinia viral core inhibits Met-tRNAf·40S initiation complex formation with physiological mRNAs

Vaccinia viral core inhibits protein synthesis in reticulocyte lysates. In partial reactions using micrococcal nuclease treated reticulocyte lysates, the viral core inhibits Met-tRNA_f binding to 40S ribosomes in response to physiological mRNAs such as globin mRNA, cowpea mosaic viral RNA, and brome mosaic viral RNA but not in response to a trinucleotide codon, AUG. The core has also no effect on Met-tRNA_f binding to 40S ribosomes in a partial reaction using partially purified peptide chain initiation factors and AUG codon.The present observation of preferential inhibition by vaccinia viral core of Met-tRNA_f·40S initiation complex formation with physiological mRNAs and not with an artificial mRNA such as AUG codon, suggests that the viral core inhibits some step(s) in peptide chain initiation involved in the recognition of structural feature(s) unique to physiological mRNAs.