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At least 19 recordsLinked to original sources
PubMed1982

Sequence of a 1.26-kb DNA fragment containing the structural gene for E.coli initiation factor IF3: presence of an AUU initiator codon.

The nucleotide sequence of a 1.26-kb pair DNA fragment containing the structural gene for Escherichia coli initiation factor IF3 has been determined. An open reading frame of 540 nucleotides is found at the position predicted by genetic studies. The amino-acid sequence deduced from the DNA sequence accounts for a molecular weight 20 530. The important feature of the coding DNA sequence is the presence of AUU as the translational initiator codon. It is 11 bases downstream of the center of a GGAGG sequence, which can strongly pair with the sequence CCUCC near the 3' terminus of 16S rRNA. The primary DNA sequence in the region of the AUU initiator codon and its role in compensating a reduced codon-anticodon interaction in initiation complex formation are discussed.

Amino Acid Sequence↗
PubMed1987

The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coli.

The expression of infC, the structural gene for translational initiation factor IF3, has been studied in different constructs under the control of the lambda PL and tac promoters. The amount of synthesized IF3 has been determined by a quantitative functional test and the levels of IF3-specific mRNA have been estimated. The synthesis of IF3 is strongly enhanced when the unusual AUU initiation codon is changed to AUG by site-directed mutagenesis. Removal of the sequence upstream from the start codon including most of the Shine-Dalgarno sequence, as well as part of a 10 bp region with potential complementarity to an internal region of the 16S rRNA, which is unique to the IF3 mRNA, reduced but did not completely abolish the high expression of infC obtained after introduction of the AUG initiation codon. The level of IF3 mRNA was found to be positively influenced by the presence of the rplT gene in the plasmid downstream from the infC gene. In vivo accumulation of a large excess of IF3, obtained when the infC gene was placed under the control of an incompletely repressed tac promoter, was not accompanied by any noticeable adverse phenotype.

Base Sequence↗
PubMed1991

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(fMet). 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(fMet). 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(fMet) and with tRNA(fMet), fMet-tRNA(fMet) 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(fMet) or tRNA(fMet) 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.

Binding Sites↗
PubMed1984

The rate of evolutionary divergence of initiation factors IF2 and IF3 in various bacterial species determined quantitatively by immunoblotting.

Antibodies to Escherichia coli translational initiation factors IF2 and IF3 were used for an immunological comparison of unpurified proteins from the following genera: Salmonella, Serratia, Proteus, Aeromonas, Pseudomonas, Streptococcus, Sarcina and Bacillus. Immunological relatedness was compared by Ouchterlony double diffusion experiments and immunoblotting analysis. Immunoblotting is a quantitative technique for measuring levels of specific proteins in crude cell lysates. We have used this technique to measure immunological distance with the assumption that the levels of the various translational components are essentially the same in the different bacterial cells examined. Both immunodiffusion and immunoblotting analysis showed a similar evolutionary relationship between the various species for the two initiation factors examined: (Escherichia = Salmonella greater than Serratia greater than Proteus greater than Aeromonas greater than Pseudomonas). Little or no crossreactivity was found using either analysis with genera: Streptococcus, Sarcina and Bacillus. Using the immunoblot distance, the two initiation factors were shown to diverge at similar rates. One advantage the immunoblotting analysis has over other immunological techniques is that the antigens can be analyzed structurally. We found, for example, that the two forms of IF2 were present in all bacterial species which cross-reacted with anti-IF2, suggesting that both forms are functionally important. Because of its sensitivity, the immunoblot analysis may be more useful than other immunological techniques in studying species that are more distantly related.

Antibodies, Bacterial↗
PubMed1984

The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria.

A 1.1-kb Hp alpha I fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.

Base Sequence↗
PubMed1985

Salt-dependent binding of Escherichia coli initiation factor 3 to nucleic acids determined by sedimentation partition chromatography.

The binding of 14CH3- initiation factor 3 (IF3) to polynucleotides is strongly dependent upon the concentration of added salt. The observed association constant, Kobs, increases by ca. a factor of 10(2) when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to all nucleic acids examined. This salt-dependent binding suggests that at physiological salt concentrations the formation of an IF3-polynucleotide complex is primarily driven by the release of cations from the nucleic acid, although anion effects are involved also. For single-stranded nucleic acids, nonelectrostatic interactions may contribute a factor of 10(2) to the value of Kobs, although accurate assessment of these interactions is complicated by anion effects. The binding of 14CH3-IF3 to the double helix, poly(A).poly(U), appears to be exclusively electrostatic. 14CH3-IF3 forms a maximum of 8 +/- 2 ion pairs with most single-stranded polynucleotides. The value of Kobs increases from ca. 10(3) to 10(5) M-1 when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to poly(A), poly(C), poly(U), and poly(A).poly(U). At physiological salt concentrations, IF3 shows no preference for any of these bases or for single or double-stranded structures. However, 14CH3-IF3 binds ca. 60 times greater to poly(A,G), at al NaCl concentrations examined, than to the other nucleic acids, indicating that IF3 has some preference for guanine-containing polynucleotides. The presence of 10 mM Mg2+ tends to reduce the value of Kobs at any given NaCl concentration, but to a smaller degree than predicted by simply a competition between Mg2+ and IF3 for the nucleic acid lattice.

Chromatography↗
PubMed1985

A fluorescence study of the binding of poly(1,N6-ethenoadenylic acid) to Escherichia coli initiation factor 3.

The binding of initiation Factor 3 (IF3) to poly (1,N6-ethenoadenylic acid) [poly(epsilon A)] was investigated by fluorescence spectroscopy. At low salt concentrations, IF3 evokes an increase in the fluorescence intensity of poly(epsilon A) due to the unstacking of the nucleotide bases. The poly(epsilon A) fluorescence enhancement titrates to an endpoint of 13 +/- 2 nucleotide residues per IF3. The maximum poly(epsilon A) fluorescence enhancement, at lattice saturation, decreases with increasing salt concentration. Even though IF3 does not produce a large fluorescence increase between 75 and 200 mM NaCl concentration, the protein still binds to poly(epsilon A) at these salt concentrations as measured by sedimentation partition chromatography; the value of Kobs for the IF3-poly(epsilon A) interaction is comparable to that of other synthetic polynucleotides. The binding of IF3 to poly(A) at 150 and 200 mM NaCl induces an increase in nucleotide base-base separation as determined by CD, yet IF3-induced disruption of base stacking of poly(epsilon A) at these same salt concentrations is not detected by fluorescence. It is likely that IF3 binds primarily to the phosphate backbone of poly(epsilon A) at low salt concentrations, producing an increase in the fluorescence intensity. But, at higher salt concentrations, the aromatic amino acids intercalate between the nucleotide bases quenching the poly(epsilon A) fluorescence.

Circular Dichroism↗
PubMed1987

The NH2-terminal cleavage of Escherichia coli translational initiation factor IF3. A mechanism to control the intracellular level of the factor?

A short form of Escherichia coli translational initiation factor IF3, repeatedly found both in vivo and in vitro, lacking the positively charged N-terminal hexapeptide has been produced by mild trypsinization. The properties of this short form of IF3 have been studied. Compared to the long native form of the factor, the shortened IF3 displays a markedly decreased thermal stability and affinity for the 30 S ribosomal subunit, as well as a reduced biological activity in protein synthesis. Following the loss of the N-terminal hexapeptide, a second peptide bond (Lys-90-Val-91) becomes easily accessible to proteolytic attack suggesting that formation of the short IF3 may be the first step in the physiological degradation of the factor.

Amino Acid Sequence↗
PubMed1984

Transcription of a gene cluster coding for two aminoacyl-tRNA synthetases and an initiation factor in Escherichia coli.

The alpha and beta subunits of phenylalanyl-tRNA synthetase are encoded by the pheS and pheT genes, respectively. These genes are clustered closely together with the genes for threonyl-tRNA synthetase (thrS) and translation initiation factor IF3 (infC); the gene order is thrS infC pheS pheT. We have used two methods to study the transcription pattern within this cluster. The first was the in vitro transcription of DNA restriction fragments with purified RNA polymerase, followed by fractionation of the RNA products by polyacrylamide gel electrophoresis. The second method was the mapping of promoters by means of the "abortive initiation" reaction of McClure and co-workers. This procedure consists of the incubation of RNA polymerase with DNA restriction fragments plus one nucleoside monophosphate and one [alpha-32P]nucleoside triphosphate; the polymerase synthesizes dinucleotide products of known sequence at promoter sites in the DNA. We found that transcription initiated at an internal site within infC (designated P1), and at two promoter sites between infC and pheS (designated P2 and P3). Transcription terminated at two sites about 200 nucleotides apart, located just before pheS. The initiation and termination signals were arranged so as to yield a nested set of overlapping transcripts. At the P1 promoter, transcription initiated with G-C, at P2 with A-C and sometimes A-G, and at P3 with G-U. Promoter activity was also found in a 3000-base interval that includes the start of the thrS gene; eight or nine transcripts (not mapped in detail) were observed, which started with at least four different dinucleotides. All major initiation sites in the gene cluster represented purine starts, although some pyrimidine initiation was observed in trace amounts. No promoter activity was found between pheS and pheT with either of the two techniques; this observation supports the conclusion that these genes are co-transcribed. No evidence was found for any promoter between the termination sites and the beginning of the pheS gene. It is suggested that one of the terminators is an attenuation site controlling the extension of transcription into pheS and pheT. Attenuation may explain the observed regulation of phenylalanyl-tRNA synthetase by the amino acid supply.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo.

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Escherichia coli protein synthesis initiation factor IF3 controls its own gene expression at the translational level in vivo.

Measurements of the relative synthesis rates of mRNAs transcribed from the gene (thrS) for threonyl-tRNA synthetase and the adjacent gene (infC) for initiation factor IF3 show four- to fivefold more infC mRNA than thrS mRNA in vivo, suggesting that infC expression can be controlled independently of thrS expression. S1 mapping experiments reveal the existence of two transcription initiation sites for infC mRNAs internal to the thrS structural gene. Both the mRNA measurements and the S1 mapping experiments indicate that the majority of infC transcription initiates at the infC proximal promoter. In agreement with these results, the deletion of the infC distal promoter from infC-lacZ gene fusions does not affect the expression of these gene fusions in vivo. Measurements of the relative synthesis rate of infC mRNA in vivo in infC- strains overproducing IF3 shows that infC mRNA levels are normal in these strains, thus suggesting that IF3 regulates the translation of infC mRNAs in vivo. Extension of these experiments using infC-lacZ gene fusions carried on lambda bacteriophage and integrated at the lambda att site on the Escherichia coli chromosome shows that the expression of infC-lacZ protein fusions, but not infC-lacZ operon fusions, is derepressed in two infC- strains. A cellular excess of IF3 represses the expression of an infC-lacZ protein fusion but not an infC-lacZ operon fusion. Measurements of the relative mRNA synthesis rates of hybrid infC-lacZ mRNA synthesized from an infC-lacZ protein fusion under conditions of a fourfold derepression or a threefold repression of hybrid IF3-beta-galactosidase expression shows that the hybrid infC-lacZ mRNA levels remain unchanged. These results indicate that the cellular levels of IF3 negatively regulate the expression of its own gene, infC, at the translational level in vivo.

Escherichia coli↗
PubMed1992

Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20.

The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron (rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would base-pair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated, i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.

Bacterial Proteins↗
PubMed1986

Formylmethionyl-tRNA binding to 30 S ribosomes programmed with homopolynucleotides and the effect of translational initiation factor 3.

Binding of the polynucleotides poly(U), poly(X) and poly(dT) to 30 S ribosomes of Escherichia coli triggers IF2-dependent binding of initiator-tRNA (fMet-tRNA) to these particles. Poly(A) and poly(C) are inactive. A minimum chain-length of approximately 100 residues in poly(U) is required for full activity in fMet-tRNA binding, although much shorter polymers bind tightly to 30 S particles and do stimulate the binding of acPhe-tRNA. The stimulation of fMet-tRNA binding to 30 S ribosomes is strongly reduced under conditions where the polynucleotides adopt secondary structure. Complexes containing fMet-tRNA and the non-cognate codon UUU or XXX are destabilized by IF3, whereas the formation of such a complex containing an AUG codon is slightly enhanced by the factor. Consistent with previous observations, it was found that all model initiation complexes containing acPhe-tRNA are strongly destabilized by IF3, even when the cognate codon (UUU) is present. Our results suggest that IF3 counteracts 'unnatural' initiation events in vitro and suggest a regulatory role for this factor in vivo.

Anticodon↗
PubMed1992

An inhibitor of elongation factor G (EF-G) GTPase present in the ribosome wash of Escherichia coli: a complex of initiation factors IF1 and IF3?

An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-[35S]Met-tRNAfMet to ribosomes. In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-[35S]Met-tRNAfMET/poly(A,U,G)/ribosome complexes. Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet. Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3. The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor. Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits.

Bacterial Proteins↗
PubMed1988

Transcriptional patterns for the thrS-infC-rplT operon of Escherichia coli.

The genes coding for threonyl-tRNA synthetase (thrS), translation initiation factor 3 (infC) and ribosomal protein L20 (rplT) are clustered in the Escherichia coli genome. Previous studies had suggested the possibility that the expression of these genes is coupled. The transcriptional events in this operon have now been examined by S1 nuclease mapping and promoter fusion studies. The results indicate that infC-containing mRNAs are initiated from three separate promoters. Two of these are located in the protein-coding region of thrS and one, P12, is the major promoter at all growth rates tested. In addition, there is co-transcription of thrS and infC from the thrS promoter (PT). A single promoter for thrS has been mapped approx. 170 nucleotides upstream from its translation initiation site. Another promoter has been located within the infC-coding region. It is separated from the next downstream gene, rplT, by a transcription end point. However, termination at this region is only 50-70% efficient and transcripts starting at this promoter can read through into rplT. These findings demonstrate that the pattern of transcription in this operon is highly complex and the mRNA levels for each of the genes is determined by a variety of factors, including multiple promoters, co-transcription and readthrough of transcription termination signals.

Amino Acyl-tRNA Synthetases↗
PubMed1989

Inducible high expression of the Escherichia coli infC gene subcloned behind a bacteriophage T7 promoter.

The gene for Escherichia coli translational initiation factor 3 (infC) has been inserted into an overexpression plasmid under the control of the bacteriophage T7 promoter. The infC plasmid was then used to transform a host with a chromosomal T7 RNA polymerase gene controlled by the lacUV5 promoter. Induction of T7 RNA polymerase expression in the host cells resulted in a 200-fold overexpression of infC mRNA and a 100-fold overproduction of initiation factor 3. Rapid batch purification of biologically active IF3 yielded predominantly the long form of IF3, implying that the short form is an artifact of purification by traditional methods.

Bacterial Proteins↗