Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control.

Mutations at the Escherichia coli pcnB locus reduce the copy number of ColE1-like plasmids. We isolated additional mutations in this gene and conducted a preliminary characterization of its product. F-prime elements carrying the pcnB region were constructed and used to show that the mutations were recessive. The wild-type pcnB gene was cloned into a low-copy-number plasmid, and its nucleotide sequence was determined. The sequence analysis indicated that pcnB is probably the first gene in an operon that contains one or more additional genes of unknown function. The pcnB locus should encode a polypeptide of 47,349 daltons (Da). A protein of this size was observed in minicells carrying a pcnB+ plasmid, and transposon insertions and deletions that truncated this protein generally abolished pcnB function. One exceptional transposon insertion at the promoter-distal end of the pcnB gene truncated the 47-kDa protein by about 20% but did not abolish complementation activity, indicating that the C-terminus of the PcnB product is dispensable. The deduced amino acid sequence of PcnB revealed numerous charged residues and, with 10% arginines, an overall basic character, suggesting that PcnB might interact with DNA or RNA in a structural capacity. Disruption of the pcnB gene by insertional mutagenesis caused a reduction in growth rate, indicating that PcnB has an important cellular function.     

The pcnB gene of Escherichia coli, which is required for ColE1 copy number maintenance, is dispensable.

The pcnB gene product of Escherchia coli is required for copy number maintenance of plasmids related to ColE1 and also for that of the IncFII plasmid R1. Because PcnB is similar to the tRNA-binding protein tRNA nucleotidyltransferase, we have suggested that the protein would be required only for processes in which an RNA is a prominent regulatory component. This appears to be so; strains deleted for pcnB, although defective in ColE1 and R1 plasmid maintenance, maintain the iteron-regulated plasmids F and P1 normally. We also find that strains deleted for pcnB grow normally, demonstrating that PcnB has no essential cellular role under the conditions tested and suggesting that regulation by antisense RNAs similar to RNAI has no critical role in any essential host process. We confirm by immunological tests that PcnB is likely to be the commercially available enzyme poly(A) polymerase.     

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.     

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli  ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.     

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 role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli

The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e. the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type. The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo. In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl-tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background. The same result was obtained with the rpsO gene encoding ribosomal protein S15. We also show that derepression of infCthrS, and rpsO is obtained with other ‘abnormal’ initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency. Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other ‘abnormal’ initiation codons. Under the same conditions and with the same set of ‘abnormal’ initiation codons, the repression of thrS and rpsO expression is weaker. This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3. We also studied another peculiarity of the infC message, namely the role of a GC-rich sequence located immediately downstream of the initiation codon and conserved through evolution. This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation. Unexpectedly, mutating this GC-rich sequence increases infC expression, indicating that this sequence has no enhancing role. Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC-rich sequence might pair with another region of the mRNA. On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between ‘normal’ and ‘abnormal’ initiation codons.     

The hisD-hisC gene border of the Salmonella typhimurium histidine operon

Summary We have sequenced the hisD-hisC gene border of the Salmonella typhimurium histidine operon. The translation termination codon of the hisD gene overlaps with the translation initiation codon of the hisC gene in the manner . The Shine-Dalgarno sequence of the hisC gene is contained entirely within hisD and there is no intercistronic space since all of the bases are utilized in coding. Two mutations that alter the hisD-hisC gene border are analyzed. Both mutations simultaneously abolish the termination codon of hisD and modify the initiation codon of hisC. One of the mutations changes the hisC initiation codon from AUG to AUU. The AUU codon is 10 to 20% as efficient as AUG for initiation of translation of the hisC gene. The mutant hisC ribosome binding site is compared to the ribosome binding site of the Escherichia coli infC gene which has been reported to contain an AUU initiation codon. The role of overlapping termination/initiation codons in regulating translation of polycistronic mRNAs in bacterial operons is discussed.     

Initiation of translation at AUC, AUA and AUU codons in Escherichia coli

A truncated form of the HBL murein hydrolase, encoded by the temperate bacteriophage HB-3, was cloned in a pUC-derivative and translated in Escherichia coli using AUC as start codon, as confirmed by biochemical, immunological, and N-terminal analyses. Using site-directed mutagenesis, we have changed this AUC codon into AUA, AUU and AUG codons. The relative translation efficiencies for these triplets were about 5% for AUC and AUU and 7.5% for AUA compared to that of AUG codon. In the same gene arrangement E. coli beta-galactosidase was also translated at moderate efficiency using AUC as initiator.     

An internal AUU codon initiates a smaller peptide encoded by bacteriophage T4 baseplate gene 26

Summary Bacteriophage T4 baseplate gene 26 codes for two in-frame overlapping peptides with identical C-terminal regions. By site-directed mutagenesis we have now determined that an internal AUU, codon 114 of gene 26, is used as the initiation codon for the synthesis of a smaller peptide (gp26^*). Thus gene 26^* gives rise to a peptide of 95 amino acid residues with an M_r of 10873, while the complete gene 26 encodes a peptide of 208 amino acid residues of M_r 23 880. Expression of gene 26^* is shown to depend on the RNA secondary structure in the translational initiation region of this gene.     

The initiation codon determines the efficiency but not the site of translation initiation in Chlamydomonas chloroplasts.

To study translation initiation in Chlamydomonas chloroplasts, we mutated the initiation codon AUG to AUU, ACG, ACC, ACU, and UUC in the chloroplast petA gene, which encodes cytochrome f of the cytochrome b6/f complex. Cytochrome f accumulated to detectable levels in all mutant strains except the one with a UUC codon, but only the mutant with an AUU codon grew well at 24 degrees C under conditions that require photosynthesis. Because no cytochrome f was detectable in the UUC mutant and because each mutant that accumulated cytochrome f did so at a different level, we concluded that any residual translation probably initiates at the mutant codon. As a further demonstration that alternative initiation sites are not used in vivo, we introduced in-frame UAA stop codons immediately downstream or upstream or in place of the initiation codon. Stop codons at or downstream of the initiation codon prevented accumulation of cytochrome f, whereas the one immediately upstream of the initiation codon had no effect on the accumulation of cytochrome f. These results suggest that an AUG codon is not required to specify the site of translation initiation in chloroplasts but that the efficiency of translation initiation depends on the identity of the initiation codon.     

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.     

Codon usage, transfer RNA availability and mistranslation in amino acid starved bacteria.

The fidelity of codon reading was examined in amino acid starved Escherichia coli. In one case the level of misincorporation of methionine was measured at an isoleucine residue encoded by either the commonly used AUU codon or the rarely used AUA codon. In this situation we found the frequency of methionine misincorporation to be very low and to be unaffected by the identity of the isoleucine codon. In other experiments histidine misincorporation for glutamine was measured in glutamine starved cells with normal levels of histidine-specific tRNA and cells overproducing this tRNA. Cells overproducing the tRNA had higher levels of misincorporation.     

Metabolic stability of the two forms of initiation factor IF-3 in Escherichia coli during the growth cycle.

Possible alteration in the ratio of the long and short forms of initiation factor IF-3 (FEBS Lett. 79, 264-275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319-325, 1972), the total amount of IF-3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithmic phase cells. The activity of IF-3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF-3/ribosome is maintained constant. The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth-cycle-dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se.     

The translational fidelity function of IF3 during transition from the 30 S initiation complex to the 70 S initiation complex

IF3 has a fidelity function in the initiation of translation, inducing the dissociation of fMet-tRNA(fMet) from the 30 S initiation complexes (30SIC) containing a non-canonical initiation triplet (e.g. AUU) in place of a canonical initiation triplet (e.g., AUG). IF2 has a complementary role, selectively promoting initiator tRNA binding to the ribosome. Here, we used parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in intensities of fluorophore-labeled IF2 and fMet-tRNA(fMet) to determine the effects on both 30SIC formation and 30SIC conversion to 70 S initiation complexes (70SIC) of (a) substituting AUG with AUU, and/or (b) omitting IF3, and/or (c) replacing GTP with the non-hydrolyzable analog GDPCP. We demonstrate that the presence or absence of IF3 has, at most, minor effects on the rate of 30SIC formation using either AUG or AUU as the initiation codon, and conclude that the high affinity of IF2 for both 30 S subunit and initiator tRNA overrides any perturbation of the codon-anticodon interaction resulting from AUU for AUG substitution. In contrast, replacement of AUG by AUU leads to a dramatic reduction in the rate of 70SIC formation from 30SIC upon addition of 50 S subunits. Interpreting our results in the framework of a quantitative kinetic scheme leads to the conclusion that, within the overall process of 70SIC formation, the step most affected by substituting AUU for AUG involves the conversion of an initially labile 70 S ribosome into a more stable complex. In the absence of IF3, the difference between AUG and AUU largely disappears, with each initiation codon affording rapid 70SIC formation, leading to the hypothesis that it is the rate of IF3 dissociation from the 70 S ribosome during IC70S formation that is critical to its fidelity function.     

Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes.

AGA and AGG codons for arginine are the least used codons in Escherichia coli, which are encoded by a rare tRNA, the product of the dnaY gene. We examined the positions of arginine residues encoded by AGA/AGG codons in 678 E. coli proteins. It was found that AGA/AGG codons appear much more frequently within the first 25 codons. This tendency becomes more significant in those proteins containing only one AGA or AGG codon. Other minor codons such as CUA, UCA, AGU, ACA, GGA, CCC and AUA are also found to be preferentially used within the first 25 codons. The effects of the AGG codon on gene expression were examined by inserting one to five AGG codons after the 10th codon from the initiation codon of the lacZ gene. The production of beta-galactosidase decreased as more AGG codons were inserted. With five AGG codons, the production of beta-galactosidase (Gal-AGG5) completely ceased after a mid-log phase of cell growth. After 22 hr induction of the lacZ gene, the overall production of Gal-AGG5 was 11% of the control production (no insertion of arginine codons). When five CGU codons, the major arginine codon were inserted instead of AGG, the production of beta-galactosidase (Gal-CGU5) continued even after stationary phase and the overall production was 66% of the control. The negative effect of the AGG codons on the Gal-AGG5 production was found to be dependent upon the distance between the site of the AGG codons and the initiation codon. As the distance was increased by inserting extra sequences between the two codons, the production of Gal-AGG5 increased almost linearly up to 8 fold. From these results, we propose that the position of the minor codons in an mRNA plays an important role in the regulation of gene expression possibly by modulating the stability of the initiation complex for protein synthesis.     

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

Summary The expression of infC, the structural gene for translational initiation factor IF3, has been studied in different constructs under the control of the P_L 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.     

In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation

To examine normal and aberrant translation initiation in Saccharomyces cerevisiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG8m to codon 91 of the COX2 coding sequence and inserted the chimeric gene into mitochondrial DNA (mtDNA). Translation of the cox2(1-91)::ARG8m mRNA yielded a fusion protein precursor that was processed to yield wild-type Arg8p. Thus mitochondrial translation could be monitored by the ability of mutant chimeric genes to complement a nuclear arg8 mutation. As expected, translation of the cox2(1-91)::ARG8m mRNA was dependent on the COX2 mRNA-specific activator PET111. We tested the ability of six triplets to function as initiation codons in both the cox2(1-91)::ARG8m reporter mRNA and the otherwise wild-type COX2 mRNA. Substitution of AUC, CCC or AAA for the initiation codon abolished detectable translation of both mRNAs, even when PET111 activity was increased. The failure of these mutant cox2(1-91)::ARG8m genes to yield Arg8p demonstrates that initiation at downstream AUG codons, such as COX2 codon 14, does not occur even when normal initiation is blocked. Three mutant triplets at the site of the initiation codon supported detectable translation, with efficiencies decreasing in the order GUG, AUU, AUA. Increased PET111 activity enhanced initiation at AUU and AUA codons. Comparisons of expression, at the level of accumulated product, of cox2(1-91)::ARG8m and COX2 carrying these mutant initiation codons revealed that very low-efficiency translation can provide enough Cox2p to sustain significant respiratory growth, presumably because Cox2p is efficiently assembled into stable cytochrome oxidase complexes.