Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine-Dalgarno sequence in prokaryotes.
ABSTRACT: In prokaryotes, translation initiation is believed to occur through an interaction between the 3? tail of a 16S rRNA and a corresponding Shine-Dalgarno (SD) sequence in the 5? untranslated region (UTR) of an mRNA. However, some genes lack SD sequences (non-SD genes), and the fraction of non-SD genes in a genome varies depending on the prokaryotic species. To elucidate non-SD translation initiation mechanisms in prokaryotes from an evolutionary perspective, we statistically examined the nucleotide frequencies around the initiation codons in non-SD genes from 260 prokaryotes (235 bacteria and 25 archaea). We identified distinct nucleotide frequency biases upstream of the initiation codon in bacteria and archaea, likely because of the presence of leaderless mRNAs lacking a 5? UTR. Moreover, we observed overall similarities in the nucleotide patterns between upstream and downstream regions of the initiation codon in all examined phyla. Symmetric nucleotide frequency biases might facilitate translation initiation by preventing the formation of secondary structures around the initiation codon. These features are more prominent in species' genomes that harbor large fractions of non-SD sequences, suggesting that a reduced stability around the initiation codon is important for efficient translation initiation in prokaryotes.
Project description:Understanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame. In this review, we focus on the comparison of start codon selection mechanisms in eukaryotes and archaea. Eukaryotic translation initiation is a very complicated process, involving many initiation factors. The most widespread mechanism for the discovery of the start codon is the scanning of the mRNA by a pre-initiation complex until the first AUG codon in a correct context is found. In archaea, long-range scanning does not occur because of the presence of Shine-Dalgarno (SD) sequences or of short 5' untranslated regions. However, archaeal and eukaryotic translation initiations have three initiation factors in common: e/aIF1, e/aIF1A and e/aIF2 are directly involved in the selection of the start codon. Therefore, the idea that these archaeal and eukaryotic factors fulfill similar functions within a common structural ribosomal core complex has emerged. A divergence between eukaryotic and archaeal factors allowed for the adaptation to the long-range scanning process versus the SD mediated prepositioning of the ribosome.
Project description:Translation generally initiates with the AUG codon. While initiation at GUG and UUG is permitted in prokaryotes (Archaea and Bacteria), cases of CUG initiation were recently reported in human cells. The varying stringency in translation initiation between eukaryotic and prokaryotic domains largely stems from a fundamental problem for the ribosome in recognizing a codon at the peptidyl-tRNA binding site. Initiation factors specific to each domain of life evolved to confer stringent initiation by the ribosome. The mechanistic basis for high accuracy in eukaryotic initiation is described based on recent findings concerning the role of the multifactor complex (MFC) in this process. Also discussed are whether non-AUG initiation plays any role in translational control and whether start codon accuracy is regulated in eukaryotes.
Project description:Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.
Project description:In prokaryotes, Shine-Dalgarno (SD) sequences, nucleotides upstream from start codons on messenger RNAs (mRNAs) that are complementary to ribosomal RNA (rRNA), facilitate the initiation of protein synthesis. The location of SD sequences relative to start codons and the stability of the hybridization between the mRNA and the rRNA correlate with the rate of synthesis. Thus, accurate characterization of SD sequences enhances our understanding of how an organism's transcriptome relates to its cellular proteome. We implemented the Individual Nearest Neighbor Hydrogen Bond model for oligo-oligo hybridization and created a new metric, relative spacing (RS), to identify both the location and the hybridization potential of SD sequences by simulating the binding between mRNAs and single-stranded 16S rRNA 3' tails. In 18 prokaryote genomes, we identified 2,420 genes out of 58,550 where the strongest binding in the translation initiation region included the start codon, deviating from the expected location for the SD sequence of five to ten bases upstream. We designated these as RS+1 genes. Additional analysis uncovered an unusual bias of the start codon in that the majority of the RS+1 genes used GUG, not AUG. Furthermore, of the 624 RS+1 genes whose SD sequence was associated with a free energy release of less than -8.4 kcal/mol (strong RS+1 genes), 384 were within 12 nucleotides upstream of in-frame initiation codons. The most likely explanation for the unexpected location of the SD sequence for these 384 genes is mis-annotation of the start codon. In this way, the new RS metric provides an improved method for gene sequence annotation. The remaining strong RS+1 genes appear to have their SD sequences in an unexpected location that includes the start codon. Thus, our RS metric provides a new way to explore the role of rRNA-mRNA nucleotide hybridization in translation initiation.
Project description:Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine- Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (-1 position) and the nucleotide 3' to the tRNAf(Met) anticodon, at position 37. Adenine is conserved at position 37, and a uridine at -1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA -1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA(fMet). The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added specificity to transcripts carrying non-standard initiation codons, and/or preserve translational fidelity under certain stress conditions.
Project description:Despite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E. coli genes, we show that interactions between the coding region and the upstream Shine-Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E. coli. We further demonstrate that far-downstream mutations can also modulate mRNA levels by occluding the SD sequence through the formation of non-equilibrium secondary structures. By contrast, a non-endogenous RNA polymerase that decouples transcription and translation largely alleviates the effects of synonymous substitutions on mRNA levels. Finally, a complementary statistical analysis of the E. coli genome specifically implicates avoidance of intra-molecular base pairing with the SD sequence. Our results provide general physical insights into the coding-level features that optimize protein expression in prokaryotes.
Project description:<h4>Background</h4>Codon pair usage (codon context) is a species specific gene primary structure feature whose evolutionary and functional roles are poorly understood. The data available show that codon-context has direct impact on both translation accuracy and efficiency, but one does not yet understand how it affects these two translation variables or whether context biases shape gene evolution.<h4>Methodologies/principal findings</h4>Here we study codon-context biases using a set of 72 orthologous highly conserved genes from bacteria, archaea, fungi and high eukaryotes to identify 7 distinct groups of codon context rules. We show that synonymous mutations, i.e., neutral mutations that occur in synonymous codons of codon-pairs, are selected to maintain context biases and that non-synonymous mutations, i.e., non-neutral mutations that alter protein amino acid sequences, are also under selective pressure to preserve codon-context biases.<h4>Conclusions</h4>Since in vivo studies provide evidence for a role of codon context on decoding fidelity in E. coli and for decoding efficiency in mammalian cells, our data support the hypothesis that, like codon usage, codon context modulates the evolution of gene primary structure and fine tunes the structure of open reading frames for high genome translational fidelity and efficiency in the 3 domains of life.
Project description:<h4>Background</h4>Shine-Dalgarno (SD) signal has long been viewed as the dominant translation initiation signal in prokaryotes. Recently, leaderless genes, which lack 5'-untranslated regions (5'-UTR) on their mRNAs, have been shown abundant in archaea. However, current large-scale in silico analyses on initiation mechanisms in bacteria are mainly based on the SD-led initiation way, other than the leaderless one. The study of leaderless genes in bacteria remains open, which causes uncertain understanding of translation initiation mechanisms for prokaryotes.<h4>Results</h4>Here, we study signals in translation initiation regions of all genes over 953 bacterial and 72 archaeal genomes, then make an effort to construct an evolutionary scenario in view of leaderless genes in bacteria. With an algorithm designed to identify multi-signal in upstream regions of genes for a genome, we classify all genes into SD-led, TA-led and atypical genes according to the category of the most probable signal in their upstream sequences. Particularly, occurrence of TA-like signals about 10 bp upstream to translation initiation site (TIS) in bacteria most probably means leaderless genes.<h4>Conclusions</h4>Our analysis reveals that leaderless genes are totally widespread, although not dominant, in a variety of bacteria. Especially for Actinobacteria and Deinococcus-Thermus, more than twenty percent of genes are leaderless. Analyzed in closely related bacterial genomes, our results imply that the change of translation initiation mechanisms, which happens between the genes deriving from a common ancestor, is linearly dependent on the phylogenetic relationship. Analysis on the macroevolution of leaderless genes further shows that the proportion of leaderless genes in bacteria has a decreasing trend in evolution.
Project description:<h4>Background</h4>Recent studies have demonstrated a selection pressure for reduced mRNA secondary-structure stability near the start codon of coding sequences. This selection pressure can be observed in bacteria, archaea, and eukaryotes, and is likely caused by the requirement of efficient translation initiation in cellular organism.<h4>Results</h4>Here, we surveyed the complete genomes of 650 dsDNA virus strains for signals of reduced stability of mRNA secondary structure near the start codon. Our analysis included viruses infecting eukaryotic, prokaryotic, and archaeic hosts. We found that many viruses showed evidence for reduced mRNA secondary-structure stability near the start codon. The effect was most pronounced in viruses infecting prokaryotes, but was also observed in viruses infecting eukaryotes and archaea. The reduction in stability generally increased with increasing genomic GC content. For bacteriophage, the reduction was correlated with a corresponding reduction of stability in the phage hosts.<h4>Conclusions</h4>We conclude that reduced stability of the mRNA secondary structure near the start codon is a common feature for dsDNA viruses, likely driven by the same selective pressures that cause it in cellular organisms.
Project description:Efficient and accurate protein synthesis is crucial for organismal survival in competitive environments. Translation efficiency-the number of proteins translated from a single mRNA in a given time period-is the combined result of differential translation initiation, elongation, and termination rates. Previous research identified the Shine-Dalgarno (SD) sequence as a modulator of translation initiation in bacterial genes, while codon usage biases are frequently implicated as a primary determinant of elongation rate variation. Recent studies have suggested that SD sequences within coding sequences may negatively affect translation elongation speed, but this claim remains controversial. Here, we present a metric to quantify the prevalence of SD sequences in coding regions. We analyze hundreds of bacterial genomes and find that the coding sequences of highly expressed genes systematically contain fewer SD sequences than expected, yielding a robust correlation between the normalized occurrence of SD sites and protein abundances across a range of bacterial taxa. We further show that depletion of SD sequences within ribosomal protein genes is correlated with organismal growth rates, supporting the hypothesis of strong selection against the presence of these sequences in coding regions and suggesting their association with translation efficiency in bacteria.