Stop Codon Context Research Articles

Termination Codons and Stop Codon Context in Bacteria and Mammalian Mitochondria

ABSTRACTThe aim of this study was to analyze the frequency of occurrence of individual stop codons and combinations of stop codons with adjacent upstream and downstream triplets in 264 bacterial and 1308 mammalian mitochondrial genomes. For the purpose of this analysis a novel program (Gene Triplet Analysis) was applied. The obtained results indicate that the standard stop codon UAA is the most frequently used one (48%) in both bacteria and mitochondria. In addition, our analysis revealed 30 nonstandard translation termination codons in mitochondria. The preferential nucleotides in all three positions adjacent to the stop codons in mammalian mitochondria are A and U.

Evidence of abundant stop codon readthrough in Drosophila and other metazoa

While translational stop codon readthrough is often used by viral genomes, it has been observed for only a handful of eukaryotic genes. We previously used comparative genomics evidence to recognize protein-coding regions in 12 species of Drosophila and showed that for 149 genes, the open reading frame following the stop codon has a protein-coding conservation signature, hinting that stop codon readthrough might be common in Drosophila. We return to this observation armed with deep RNA sequence data from the modENCODE project, an improved higher-resolution comparative genomics metric for detecting protein-coding regions, comparative sequence information from additional species, and directed experimental evidence. We report an expanded set of 283 readthrough candidates, including 16 double-readthrough candidates; these were manually curated to rule out alternatives such as A-to-I editing, alternative splicing, dicistronic translation, and selenocysteine incorporation. We report experimental evidence of translation using GFP tagging and mass spectrometry for several readthrough regions. We find that the set of readthrough candidates differs from other genes in length, composition, conservation, stop codon context, and in some cases, conserved stem-loops, providing clues about readthrough regulation and potential mechanisms. Lastly, we expand our studies beyond Drosophila and find evidence of abundant readthrough in several other insect species and one crustacean, and several readthrough candidates in nematode and human, suggesting that functionally important translational stop codon readthrough is significantly more prevalent in Metazoa than previously recognized.

Correlation of mRNA Expression and Protein Abundance Affected by Multiple Sequence Features Related to Translational Efficiency in Desulfovibrio vulgaris: A Quantitative Analysis

The modest correlation between mRNA expression and protein abundance in large-scale data sets is explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Among various factors affecting the mRNA-protein correlation, the roles of biological factors related to translation are poorly understood. In this study, using experimental mRNA expression and protein abundance data collected from Desulfovibrio vulgaris by DNA microarray and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic analysis, we quantitatively examined the effects of several translational-efficiency-related sequence features on mRNA-protein correlation. Three classes of sequence features were investigated according to different translational stages: (i) initiation, Shine-Dalgarno sequences, start codon identity, and start codon context; (ii) elongation, codon usage and amino acid usage; and (iii) termination, stop codon identity and stop codon context. Surprisingly, although it is widely accepted that translation initiation is the rate-limiting step for translation, our results showed that the mRNA-protein correlation was affected the most by the features at elongation stages, i.e., codon usage and amino acid composition (5.3-15.7% and 5.8-11.9% of the total variation of mRNA-protein correlation, respectively), followed by stop codon context and the Shine-Dalgarno sequence (3.7-5.1% and 1.9-3.8%, respectively). Taken together, all sequence features contributed to 15.2-26.2% of the total variation of mRNA-protein correlation. This study provides the first comprehensive quantitative analysis of the mRNA-protein correlation in bacterial D. vulgaris and adds new insights into the relative importance of various sequence features in prokaryotic protein translation.

Cis control of gene expression in E.coli by ribosome queuing at an inefficient translational stop signal.

An UGA stop codon context which is inefficient because of the 3'-flanking context and the last two amino acids in the gene protein product has a negative effect on gene expression, as shown using a model protein A' gene. This is particularly true at low mRNA levels, corresponding to a high intracellular ribosome/mRNA ratio. The negative effect is smaller if this ratio is decreased, or if the distance between the initiation and termination signals is increased. The results suggest that an inefficient termination codon can cause ribosomal pausing and queuing along the upstream mRNA region, thus blocking translation initiation of short genes. This cis control effect is dependent on the stop codon context, including the C-terminal amino acids in the gene product, the translation initiation signal strength, the ribosome/mRNA ratio and the size of the mRNA coding region. A large proportion of poorly expressed natural Escherichia coli genes are small, and the weak termination codon UGA is under-represented in small, highly expressed E.coli genes as compared with the efficient stop codon UAA.

Impact of the six nucleotides downstream of the stop codon on translation termination.

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3' termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence -CA(A/G)N(U/C/G)A-, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3' stop codon context on translation termination.

UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals

BackgroundTranslation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.ResultsWe have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10-4. We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed.ConclusionsWe conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders.

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

TransTerm, the translational signal database, extended to include full coding sequences and untranslated regions.

TransTerm is a database of mRNA sequences and parameters useful for detecting translational control signals in general. TransTerm-98 has been expanded beyond previous years to include full coding sequences and UTRs, while retaining the original small contexts about the coding sequence start- and stop-codons. The database contains more than 130 000 non-redundant coding sequences with associated untranslated regions (UTRs) from over 450 species. This includes the complete genomes of 12 prokaryotic and one eukaryotic organism. Several coding sequence parameters are available: coding sequence length, Nc, GC3 and, when it is computable, Codon Adaptation Index (CAI). Codon usage tables and summaries of start- and stop-codon contexts are also included. TransTerm-98 has both a relational database form with a WWW interface and a flatfile format, also available by Internet browser. TransTerm is available at: http://biochem.otago.ac.nz:800/Transterm/homepage.h tml

The translational signal database, TransTerm, is now a relational database.

TransTerm-97 contains more than 97 500 non-redundant coding-sequence initiation and termination contexts compiled from GenBank, release 101 (15-June-1997). In addition, several coding sequence parameters are available: coding sequence length, Nc, GC3, and, when it is computable, codon adaptation index (CAI). Codon usage tables and summaries of start and stop codon contexts are also included. The information covers more than 325 species and organelles, including seven complete bacterial genomes and one complete eukaryotic genome. To promote research in translational control of protein synthesis, TransTerm has been converted into a relational database to ease the process of making queries. The relational database manager, Postgresql, gives access to the database using SQL (Structured Query Language). A World Wide Web interface using forms is being completed to allow the casual user access to the database. Extensions are planned to include the full 5'-UTR, full coding sequence and 3'-UTR. TransTerm-97 is available on the World Wide Web at:http://biochem. otago.ac.nz:800/Transterm/homepage.html

Release factor RF3 abolishes competition between release factor RF1 and ribosome recycling factor (RRF) for a ribosome binding site

The dependence of the rate of ribosomal recycling (from initiation via protein elongation and termination, and then back to initiation) on the concentrations of release factor RF1 and the ribosome recycling factor (RRF) has been studied in vitro. High RF1 concentration was found to reduce the rate of ribosomal recycling and the extent of this reduction depended on stop codon context. The inhibitory effect of high RF1 concentrations can be reversed by a corresponding increase in RRF concentration. This indicates that RF1 and RRF have mutually exclusive and perhaps overlapping binding sites on the ribosome. Addition of release factor RF3 to the translation system abolishes the inhibitory effect of high RF1 concentration and increases the overall rate of ribosome recycling. These data can be explained by a three-step model for termination where the first step is RF1-promoted hydrolysis of peptidyl-tRNA. The second step is an intrinsically slow dissociation of RF1 which is accelerated by RF3. The third step, catalysed by RRF and elongation factor G, leads to mobility of the ribosome on mRNA allowing it to enter a further round of translation. In the absence of RF3, RF1 can re-associate rapidly with the ribosome after peptidyl-tRNA hydrolysis, preventing RRF from entering the ribosomal A-site and thereby inhibiting ribosomal recycling. The overproduction of RF1 in cells deficient in RRF or lacking RF3 has effects on growth rate predicted by the in vitro experiments.

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.

Analysis of the stop codon context in plant nuclear genes

A region of 18 nucleotides surrounding the stop codon (the stop codon context) in 748 plant nuclear genes was analyzed. Non-randomness was found both upstream and downstream from the stop codon, suggesting that these sequences may help in ensuring efficient termination of translation. The UAG amber codon is the least-used stop codon and the bias in the nucleotide distribution 5' and 3' to the stop codon was more pronounced for the amber codon than for the other stop codons. This might indicate that the codon context affects termination more at UAG than at UGA or UAA stop codons.