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At least 19 recordsLinked to original sources
PubMed1990

Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3.

Initiation factors are used by Escherichia coli to select the initiator tRNA over elongator tRNAs during translation initiation. IF3 appears to "inspect" the anticodon end of the tRNA, probably along with the initiation codon. The anticodon stem and loop of the initiator tRNA, together with part of the initiation codon of the mRNA, can be thought of as a unit. Changes made in the anticodon stem, the anticodon loop, or the anticodon of an initiator tRNA fragment result in a loss of selection by IF3 in an in vitro assay for translation initiation. IF3 allows the selection of an initiator tRNA anticodon stem and loop fragment on GUG and UUG codons but does not select that tRNA fragment in response to AUU.

Bacterial Proteins↗
PubMed1991

Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae.

A set of 32 different codons were introduced in a lacZ expression vector (pPTK400) immediately 3' from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of beta-galactosidase fusion protein using immuno-gel electrophoresis. A 5.3-fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinants of the optimal context for an initiation codon in yeast therefore must be located mainly 5' from this codon.

Amino Acid Sequence↗
PubMed1987

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Bacteriophage lambda↗
PubMed1995

Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes.

Codon usage by all the known gene sequences from Autographa californica nuclear polyhedrosis virus (AcNPV) was compared with that of firefly luciferase (luc) and the beta subunit of human chorionic gonadotropin (beta hCG) expressed to contrasting levels in the baculovirus system. The highly expressed luc gene showed a codon usage similar to AcNPV genes, as reflected by a very low D-squared statistic value (0.78) and a similar G/C usage (45%) at wobble positions. However, the underexpressed beta hCG gene displayed a high D-squared value (7.3) and G/C usage (82.5%) at the wobble base position. Alignment of the 20 nucleotides around the initiation codon of 23 AcNPV genes identified a novel consensus translation initiation sequence aag/ta/tat/aa/cAAaATGaa/ct/ag/aAan, which was quite different from the Kozak consensus sequence (GCC)GCCA/GCCATGG. An extension of these analyses to a sample of other heterologous genes overexpressed and underexpressed in BEVS suggested similar trends. These theoretical analyses have important implications for heterologous gene expression in this system.

Animals↗
PubMed1987

Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.

Base Sequence↗
PubMed1996

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 infC, thrS, 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.

Bacterial Proteins↗
PubMed1991

Selection for a pre-C stop codon mutation in a hepatitis B virus variant with a pre-C initiation codon mutation during interferon treatment.

Hepatitis B virus (HBV) variants with a stop codon, a mutated translation initiation codon or other mutations in the pre-C region which prevent e-antigen expression are highly prevalent in anti-HBe chronic carriers and can be positively selected from a mixed virus infection. Our laboratories recently described pre-C variants with two pre-C mutations which prevent HBeAg expression. Here we have investigated whether there is a selective pressure for acquisition of the second pre-C mutation. By direct sequencing of amplified HBV DNA from sera of a chronic carrier taken during a 6-year follow-up, we found that genomes of a virus population virtually all had a pre-C translation initiation codon mutation and about 50% had an additional stop codon mutation. With the onset of interferon treatment, the genomes with the stop codon mutation increased to more than 95% while the frequency of the translation initiation codon mutation in all genomes remained constant. These data indicate positive selection (possibly immune-mediated and HBeAg-targeted) for a second pre-C mutation. This putative enhancement of negative translational control may be present because a pre-C translation initiation codon mutation cannot totally prevent HBeAg expression and is therefore less frequent.

Adult↗
PubMed1995

Creation of an initiation codon by RNA editing in the coxI transcript from tomato mitochondria.

Nucleotide-sequence analysis showed that the gene for cytochrome oxidase subunit I (coxI) from tomato mitochondrial DNA has an ACG codon at a conserved position corresponding to an ATG initiation codon in other higher-plant coxI genes. cDNA-sequence analysis of the coxI transcripts showed that 15 positions in the genomic DNA were converted from C to U in the transcripts by RNA editing. One of the editing events is observed at the indicated ACG codon, producing an ATG initiation codon. The nucleotide sequences of 37 cDNA clones showed that the initiation codon was created in 32 out of the 37 clones, while nucleotide positions 254 and 11 were edited in 37 and 34 of the 37 clones examined, respectively, suggesting that creation of the initiation codon is a post-transcriptional event. The BamHI site at nucleotide position 757-762 within the coxI genomic DNA was altered in all 97 cDNA clones examined, demonstrating that RNA editing at this site in the transcripts is very common. RNA editing takes place to a lesser extent at the initiation codon, compared with editing at internal position 254. This indicates that editing is either a random process or that it involves a mechanism favoring less RNA editing in the initiation codon than in internal sites.

Amino Acid Sequence↗
PubMed1980

AUG is the only initiation codon in eukaryotes.

An analysis of mutants of the yeast Saccharomyces cerevisiae indicates that AUG is the sole codon capable of initiating translation of iso-1-cytochrome c. This result with yeast and the sequence results of numerous eukaryotic genes indicate that AUG is the only initiation codon in eukaryotes; in contrast, results with Escherichia coli and bacteriophages indicate that both AUG and GUG are initiation codons in prokaryotes. The difference can be explained by the lack of the t6 A hypermodified nucleoside (N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine) in prokaryotic initiator tRNA and its presence in eukaryotic initiator tRNA.

Cells↗
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↗
PubMed1995

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.

Animals↗
PubMed1997

Functional importance of RNA interactions in selection of translation initiation codons.

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA-small ribosomal subunit-initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis-acting mRNA elements and trans-acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.

Base Sequence↗
PubMed1992

The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.

Animals↗
PubMed1989

Translational regulation of the lysis gene in RNA bacteriophage fr requires a UUG initiation codon.

Single nucleotide substitutions identify a UUG triplet as the initiation codon of the lysis gene in RNA bacteriophage fr. This initiation codon is non-functional in de novo initiation but is activated by translational termination at the overlapping coat gene. The UUG initiation codon is crucial for gene regulation in the phage, as it excludes uncontrolled access of ribosomes to the start of the lysis gene. Replacement of UUG by either GUG or AUG results in the loss of genetic control of the lysis gene. A model is presented in which initiation factor IF3 proofreads de novo initiation at UUG codons.

Bacteriolysis↗
PubMed2026

A-to-I RNA editing remodels 5'-UTR initiation codons to tune translational output.

A-to-I RNA editing is a prevalent post-transcriptional modification in higher eukaryotes that converts adenosine to inosine within RNA molecules. Because inosine is interpreted as guanosine during translation, editing can alter codon identity and potentially influence translation initiation signals. Here, we examined whether A-to-I editing within the 5' untranslated region (5'-UTR) can remodel upstream initiation codons and thereby tune downstream translation. Using luciferase-based reporter systems, we show that AUA-to-AUI editing generates an initiation-competent inosine-containing codon, whereas AUG-to-IUG editing markedly attenuates initiation and can relieve uORF-mediated repression. Quantitative in vitro and cellular assays establish the initiation hierarchy AUA&#x2009;<&#x2009;AUI&#x2009;<&#x2009;AUG, with IUG exhibiting strongly reduced initiation efficiency. Importantly, AUI-mediated upstream initiation did not behave like a canonical AUG-initiated uORF in the tested contexts; its effect on downstream ORF translation was modest and context-dependent. Transcriptome-wide bioinformatic analysis identified endogenous human transcripts whose 5'-UTRs harbor editing sites compatible with initiation-codon gain or attenuation. Reporter validation using native 5'-UTR sequences supports the possibility that editing-dependent initiation-codon remodeling can tune translational output in living cells, particularly through AUG-to-IUG-mediated derepression. Together, these findings establish a reporter-based framework in which A-to-I editing can remodel 5'-UTR initiation codons, while highlighting the need for endogenous protein-level and native-locus validation to determine physiological relevance.

RNA Editing↗
PubMed1986

Oligonucleotide directed mutagenesis of cauliflower mosaic virus DNA using a repair-resistant nucleoside analogue: identification of an agnogene initiation codon.

Mutation of the initiation codon of the dispensible open reading frame, ORF VII, of cauliflower mosaic virus (CaMV) delayed the appearance of disease symptoms, but the mutants reverted with high frequency. This suggests a role of this start codon in viral expression. Oligonucleotide-directed mutagenesis, utilizing a novel, repair-resistant deoxyguanosine analogue, 2'-deoxy-7-deazainosine (dDI), highly improved the yield of mutants.

Base Sequence↗
PubMed1988

Effect of structure of the initiator codon on translation in E. coli.

A set of plasmids carrying different initiator codons--either AUG, or GUG, or UUG, or CUG (as a control) in the hybrid gene lacIZ--was constructed by using synthetic oligonucleotides. GUG and UUG codons were demonstrated to be 2-3 times less effective than AUG in translation initiation. Furthermore, the correlation between the efficiencies of different initiator codons in translation initiation proved to vary, depending on the phase of bacterial growth. The rarely occurring usage in nature of the initiator codons GUG and UUG is supposed to be due to the particular role played by the initiator triplets in regulation of gene expression.

Bacteriophage lambda↗