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Global fitness landscapes of the Shine-Dalgarno sequence.

ABSTRACT: Shine-Dalgarno sequences (SD) in prokaryotic mRNA facilitate protein translation by pairing with rRNA in ribosomes. Although conventionally defined as AG-rich motifs, recent genomic surveys reveal great sequence diversity, questioning how SD functions. Here, we determined the molecular fitness (i.e., translation efficiency) of 49 synthetic 9-nt SD genotypes in three distinct mRNA contexts in Escherichia coli We uncovered generic principles governing the SD fitness landscapes: (1) Guanine contents, rather than canonical SD motifs, best predict the fitness of both synthetic and endogenous SD; (2) the genotype-fitness correlation of SD promotes its evolvability by steadily supplying beneficial mutations across fitness landscapes; and (3) the frequency and magnitude of deleterious mutations increase with background fitness, and adjacent nucleotides in SD show stronger epistasis. Epistasis results from disruption of the continuous base pairing between SD and rRNA. This "chain-breaking" epistasis creates sinkholes in SD fitness landscapes and may profoundly impact the evolution and function of prokaryotic translation initiation and other RNA-mediated processes. Collectively, our work yields functional insights into the SD sequence variation in prokaryotic genomes, identifies a simple design principle to guide bioengineering and bioinformatic analysis of SD, and illuminates the fundamentals of fitness landscapes and molecular evolution.

SUBMITTER: Kuo ST

PROVIDER: S-EPMC7263185 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Predicting Shine-Dalgarno sequence locations exposes genome annotation errors.

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.

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The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria.

Project description:Protein synthesis by ribosomes takes place on a linear substrate but at non-uniform speeds. Transient pausing of ribosomes can affect a variety of co-translational processes, including protein targeting and folding. These pauses are influenced by the sequence of the messenger RNA. Thus, redundancy in the genetic code allows the same protein to be translated at different rates. However, our knowledge of both the position and the mechanism of translational pausing in vivo is highly limited. Here we present a genome-wide analysis of translational pausing in bacteria by ribosome profiling--deep sequencing of ribosome-protected mRNA fragments. This approach enables the high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare transfer RNAs do not lead to slow translation under nutrient-rich conditions. Instead, Shine-Dalgarno-(SD)-like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome possessing an altered anti-SD sequence, we show that pausing is due to hybridization between the mRNA and 16S ribosomal RNA of the translating ribosome. In protein-coding sequences, internal SD sequences are disfavoured, which leads to biased usage, avoiding codons and codon pairs that resemble canonical SD sites. Our results indicate that internal SD-like sequences are a major determinant of translation rates and a global driving force for the coding of bacterial genomes.

| S-EPMC3338875 | biostudies-literature

Re-annotation of 12,495 prokaryotic 16S rRNA 3' ends and analysis of Shine-Dalgarno and anti-Shine-Dalgarno sequences.

Project description:We examined 20,648 prokaryotic unique taxids with respect to the annotation of the 3' end of the 16S rRNA, which contains the anti-Shine-Dalgarno sequence. We used the sequence of highly conserved helix 45 of the 16S rRNA as a guide. By this criterion, 8,153 annotated 3' ends correctly included the anti-Shine-Dalgarno sequence, but 12,495 were foreshortened or otherwise mis-annotated, missing part or all of the anti-Shine-Dalgarno sequence, which immediately follows helix 45. We re-annotated, giving a total of 20,648 16S rRNA 3' ends. The vast majority indeed contained a consensus anti-Shine-Dalgarno sequence, embedded in a highly conserved 13 base "tail". However, 128 exceptional organisms had either a variant anti-Shine-Dalgarno, or no recognizable anti-Shine-Dalgarno, in their 16S rRNA(s). For organisms both with and without an anti-Shine-Dalgarno, we identified the Shine-Dalgarno motifs actually enriched in front of each organism's open reading frames. This showed to what extent the Shine-Dalgarno motifs correlated with anti-Shine Dalgarno motifs. In general, organisms whose rRNAs lacked a perfect anti-Shine-Dalgarno motif also lacked a recognizable Shine-Dalgarno. For organisms whose 16S rRNAs contained a perfect anti-Shine-Dalgarno motif, a variety of results were obtained. We found one genus, Alteromonas, where several taxids apparently maintain two different types of 16S rRNA genes, with different, but conserved, antiSDs. The fact that some organisms do not seem to have or use Shine-Dalgarno motifs supports the idea that prokaryotes have other robust mechanisms for recognizing start codons for translation.

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Selection of Shine-Dalgarno sequences in plastids.

Project description:Like bacterial genes, most plastid (chloroplast) genes are arranged in operons and transcribed as polycistronic mRNAs. Plastid protein biosynthesis occurs on bacterial-type 70S ribosomes and translation initiation of many (but not all) mRNAs is mediated by Shine-Dalgarno (SD) sequences. To study the mechanisms of SD sequence recognition, we have analyzed translation initiation from mRNAs containing multiple SD sequences. Comparing translational efficiencies of identical transgenic mRNAs in Escherichia coli and plastids, we find surprising differences between the two systems. Most importantly, while internal SD sequences are efficiently recognized in E. coli, plastids exhibit a bias toward utilizing predominantly the 5'-most SD sequence. We propose that inefficient recognition of internal SD sequences provides the raison d'être for most plastid polycistronic transcripts undergoing post-transcriptional cleavage into monocistronic mRNAs.

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2012-04-01 | E-GEOD-35641 | biostudies-arrayexpress

The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts.

Project description:In response to intracellular signals in Gram--negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)-regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by 'bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.

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Accessibility of the Shine-Dalgarno Sequence Dictates N-Terminal Codon Bias in E. coli.

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.

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Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome.

Project description:Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.

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