IF3-mediated suppression of a GUA initiation codon mutation in the recJ gene of Escherichia coli.

A mutational change of the initiation codon to GUA was found to reduce, but not abolish, expression of the recJ gene of Escherichia coli. Specific mutations in translational initiation factor IF3 have been isolated as second-site suppressors of this GUA initiation codon mutation. One of these, infC135, with an arginine-to-proline change at amino acid 131, completely restores a wild-type phenotype to recJ GUA initiation codon mutants and acts in a semidominant fashion. The infC135 mutation increased expression of RecJ from the GUA mutant but had no effect on the normal GUG start. The infC135 mutation also abolished autoregulation of IF3 in cis and in trans. The behavior of this IF3 mutant suggests that it has specifically lost its ability to abort initiation from poor initiation codons such as GUA of recJ and the AUU of infC. Because of the impact of IF3 on recJ, a recombination and repair gene, this role of IF3 must be general and not restricted to translation genes. The dominance of infC135 suggests that the other functions of IF3, for instance its ability to bind to 30S ribosomes, must remain intact. Although the ability to discriminate among initiation codons has been lost in the infC135 mutant, translational initiation was still restricted to the normal initiation site in recJ, even in the presence of a closely juxtaposed alternative initiation codon. Because the recJ gene lacks a canonical Shine-Dalgarno sequence, other unknown features of the mRNA must serve to specify the initiation site.     

The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo.

The amino acid sequence of the Dsg protein is 50% identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene. Anti-E. coli IF3 antibodies cross-react with the Dsg protein. Tn5 insertion mutations in dsg are lethal. When ample nutrients are available, however, certain dsg point mutant strains grow at the same rate as wild-type cells. Under the starvation conditions that induce fruiting body development, these dsg mutants begin to aggregate but fail to develop further. The level of Dsg antigen, as a fraction of total cell protein, does not change detectably during growth and development, as expected for a factor essential for protein synthesis. The amount of IF3 protein in E. coli is known to be autoregulated at the translational level. This autoregulation is lost in an E. coli infC362 missense mutant. The dsg+ gene from Myxococcus xanthus restores normal autoregulation to the infC362 mutant strain. Dsg is distinguished from IF3 of E. coli, other enteric bacteria, and Bacillus stearothermophilus by having a C-terminal tail of 66 amino acids. Partial and complete deletion of this tail showed that it is needed for certain vegetative and developmental functions but not for viability.     

Mutations that alter initiation codon discrimination by Escherichia coli initiation factor IF3.

This work describes the isolation of mutations in infC, the structural gene for IF3, using different genetic screens. Among 21 mutants characterised, seven were shown to produce stable variant IF3 proteins unable to fully complement a strain carrying a chromosomal deletion of the infC gene. The mutants were also shown to be unable to normally discriminate against several non-canonical initiation codons such as AUU and ACG. The two mutants with the strongest complementation or discrimination defects carry changes in the C-terminal domain of IF3, which is responsible for the binding of the factor to the 30 S ribosomal subunit. We show that the first mutant has an expected decreased but the second an unexpected increased capacity to bind the 30 S subunit. The in vivo defects of the second mutant are explained by its capacity to bind unspecifically to other targets, as shown by its increased affinity for the 50 S subunit, which is normally not recognised by the factor. Interestingly, this mutant corresponds to a change of an acidic residue that might play a negative discriminatory role in preventing interactions with non-cognate RNAs, as has been reported for acidic residues of aminoacyl-tRNA synthetases shown to be involved in tRNA recognition.     

Computational exploration of structural hypotheses for an additional sequence in a mammalian mitochondrial protein

Background

Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2_mt) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2_mt to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.

Results

The present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2_mt using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2_mt predictions differ from the observed IF2_mt cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed.

Conclusions

By hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2_mt models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2_mt cryo-EM map to get reasonable quasi-atomic IF2_mt models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2_mt models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine.     

Evidence for a role of initiation factor 3 in recycling of ribosomal complexes stalled on mRNAs in Escherichia coli

Specific interactions between ribosome recycling factor (RRF) and elongation factor-G (EFG) mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EFG are also important in peptidyl-tRNA release from stalled pre-termination complexes. Unlike the post-termination complexes (harboring deacylated tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EFG in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, (i) Inclusion of IF3 with RRF and EFG results in recycling of the pre-termination complexes; (ii) IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (iii) Transduction of infC135 (which encodes a functionally compromised IF3) in E.coli LJ14 generates a ‘synthetic severe’ phenotype; (iv) The infC135 and frr1 (containing an insertion in the RRF gene promoter) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E.coli; and (v) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E.coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E.coli.     

Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene ( tnp ), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se , was not increased. Rather, these mutations appear to increase expression of the tnp'–'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo : Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1–3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification. Importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3–5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo , consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.     

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation inEscherichia coli

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.     

Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless λ c I and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine–Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.     

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation inEscherichia coli

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.     

Multicopy suppression of a gacA mutation by the infC operon in Pseudomonas fluorescens CHA0: competition with the global translational regulator RsmA

The gacA gene of the biocontrol strain Pseudomonas fluorescens CHA0 codes for a response regulator which, together with the sensor kinase GacS (=LemA), is required for the production of exoenzymes and secondary metabolites involved in biocontrol, including hydrogen cyanide (HCN). A gacA multicopy suppressor was isolated from a cosmid library of strain CHA0 and identified as the infC-rpmI-rplT operon, which encodes the translation initiation factor IF3 and the ribosomal proteins L35 and L20. The efficiency of suppression was about 30%, as determined by the use of a GacA-controlled reporter construct, i.e. a translational hcnA'-'lacZ fusion. Overexpression of the rsmA gene (coding for a global translational repressor) reversed the suppressive effect of the amplified infC operon. This finding suggests that some product(s) of the infC operon can compete with RsmA at the level of translation in P. fluorescens CHA0 and that important biocontrol traits can be regulated at this level.