anti-SD Sequence Research Articles

Differential Selection for Translation Efficiency Shapes Translation Machineries in Bacterial Species.

Different bacterial species have dramatically different generation times, from 20-30 min in Escherichia coli to about two weeks in Mycobacterium leprae. The translation machinery in a cell needs to synthesize all proteins for a new cell in each generation. The three subprocesses of translation, i.e., initiation, elongation, and termination, are expected to be under stronger selection pressure to optimize in short-generation bacteria (SGB) such as Vibrio natriegens than in the long-generation Mycobacterium leprae. The initiation efficiency depends on the start codon decoded by the initiation tRNA, the optimal Shine-Dalgarno (SD) decoded by the anti-SD (aSD) sequence on small subunit rRNA, and the secondary structure that may embed the initiation signals and prevent them from being decoded. The elongation efficiency depends on the tRNA pool and codon usage. The termination efficiency in bacteria depends mainly on the nature of the stop codon and the nucleotide immediately downstream of the stop codon. By contrasting SGB with long-generation bacteria (LGB), we predict (1) SGB to have more ribosome RNA operons to produce ribosomes, and more tRNA genes for carrying amino acids to ribosomes, (2) SGB to have a higher percentage of genes using AUG as the start codon and UAA as the stop codon than LGB, (3) SGB to exhibit better codon and anticodon adaptation than LGB, and (4) SGB to have a weaker secondary structure near the translation initiation signals than LGB. These differences between SGB and LGB should be more pronounced in highly expressed genes than the rest of the genes. We present empirical evidence in support of these predictions.

Optimizing Protein Production in Therapeutic Phages against a Bacterial Pathogen, Mycobacterium abscessus

Therapeutic phages against pathogenic bacteria should kill the bacteria efficiently before the latter evolve resistance against the phages. While many factors contribute to phage efficiency in killing bacteria, such as phage attachment to host, delivery of phage genome into the host, phage mechanisms against host defense, phage biosynthesis rate, and phage life cycle, this paper focuses only on the optimization of phage mRNA for efficient translation. Phage mRNA may not be adapted to its host translation machinery for three reasons: (1) mutation disrupting adaptation, (2) a recent host switch leaving no time for adaptation, and (3) multiple hosts with different translation machineries so that adaptation to one host implies suboptimal adaptation to another host. It is therefore important to optimize phage mRNAs in therapeutic phages. Theoretical and practical principles based on many experiments were developed and applied to phages engineered against a drug-resistant Mycobacterium abscessus that infected a young cystic fibrosis patient. I provide a detailed genomic evaluation of the three therapeutic phages with respect to translation initiation, elongation, and termination, by making use of both experimental results and highly expressed genes in the host. For optimizing phage genes against M. abscessus, the start codon should be AUG. The DtoStart distance from base-pairing between the Shine-Dalgarno (SD) sequence and the anti-SD sequence should be 14–16. The stop codon should be UAA. If UAG or UGA is used as a stop codon, they should be followed by nucleotide U. Start codon, SD, or stop codon should not be embedded in a secondary structure that may obscure the signals and interfere with their decoding. The optimization framework should be generally applicable to developing therapeutic phages against bacterial pathogens.

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

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.

Global analysis of protein synthesis in Flavobacterium johnsoniae reveals the use of Kozak-like sequences in diverse bacteria.

In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine–Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position –3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved β-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.

Optimizing Phage Translation Initiation

Phage as an anti-bacterial agent must be efficient in killing bacteria, and consequently needs to replicate efficiently. Protein production is a limiting step in replication in almost all forms of life, including phages. Efficient protein production depends on the efficiency of translation initiation, elongation and termination, with translation initiation often being rate limiting. Initiation signals such as Shine-Dalgarno (SD) sequences and start codon are decoded by anti-SD sequences and initiation tRNA, respectively. While the decoding machinery cannot be readily modified, the signals can be engineered to increase the efficiency of their decoding. Here I review our understanding of the translation machinery to facilitate the engineering of optimal translation initiation signals for facilitating the design of phage protein-coding genes, including 1) accurate characterization of the 3’ end of 16S rRNA by using RNA-Seq data, 2) identification of the optimal SD/aSD interaction, and 3) reduction of secondary structure in sequences flanking the start codon.

Diversity of Translation Initiation Mechanisms across Bacterial Species Is Driven by Environmental Conditions and Growth Demands.

The Shine–Dalgarno (SD) sequence motif is frequently found upstream of protein coding genes and is thought to be the dominant mechanism of translation initiation used by bacteria. Experimental studies have shown that the SD sequence facilitates start codon recognition and enhances translation initiation by directly interacting with the highly conserved anti-SD sequence on the 30S ribosomal subunit. However, the proportion of SD-led genes within a genome varies across species and the factors governing this variation in translation initiation mechanisms remain largely unknown. Here, we conduct a phylogenetically informed analysis and find that species capable of rapid growth contain a higher proportion of SD-led genes throughout their genomes. We show that SD sequence utilization covaries with a suite of genomic features that are important for efficient translation initiation and elongation. In addition to these endogenous genomic factors, we further show that exogenous environmental factors may influence the evolution of translation initiation mechanisms by finding that thermophilic species contain significantly more SD-led genes than mesophiles. Our results demonstrate that variation in translation initiation mechanisms across bacterial species is predictable and is a consequence of differential life-history strategies related to maximum growth rate and environmental-specific constraints.

Elucidating the 16S rRNA 3\u2032 boundaries and defining optimal SD/aSD pairing in Escherichia coli and Bacillus subtilis using RNA-Seq data

Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5′ UTR of mRNA and the anti-SD (aSD) sequence at the free 3′ end of the 16S rRNA (3′ TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3′ TAIL and 2) extent of the core aSD sequence within the 3′ TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves 1) mapping the 3′ boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3′ TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3′ TAIL in B. subtilis and recovered the established 3′ TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein.

In Vitro Evolution of Unmodified 16S rRNA for Simple Ribosome Reconstitution.

One of the largest challenges in the synthesis of artificial cells that can reproduce is in vitro assembly of ribosomes from in vitro synthesized rRNAs and proteins. In this study, to circumvent the post-transcriptional modification of 16S rRNA for reconstitution of the fully active 30S subunit, we performed artificial evolution of 16S rRNA, which forms the functional 30S subunit without post-transcriptional modifications. We first established an in vitro selection scheme by combining the integrated synthesis, assembly, and translation (iSAT) system with the liposome sorting technique. After 15 rounds of selection cycles, we found one point mutation (U1495C) near the 3' terminus that significantly enhanced the reconstitution activity of the functional 30S subunit from unmodified 16S rRNA to approximately 57% of that from native-modified 16S rRNA. The effect of the mutation did not depend on the reconstitution scheme, anti-SD sequences, or the target genes to be translated. The mutation we found in this study enabled reconstitution of the active 30S subunit without rRNA modification, and thus would be a useful tool for simple construction of self-reproducing ribosomes.

How Changes in Anti-SD Sequences Would Affect SD Sequences in Escherichia coli and Bacillus subtilis.

The 3′ end of the small ribosomal RNAs (ssu rRNA) in bacteria is directly involved in the selection and binding of mRNA transcripts during translation initiation via well-documented interactions between a Shine-Dalgarno (SD) sequence located upstream of the initiation codon and an anti-SD (aSD) sequence at the 3′ end of the ssu rRNA. Consequently, the 3′ end of ssu rRNA (3′TAIL) is strongly conserved among bacterial species because a change in the region may impact the translation of many protein-coding genes. Escherichia coli and Bacillus subtilis differ in their 3′ ends of ssu rRNA, being GAUCACCUCCUUA3′ in E. coli and GAUCACCUCCUUUCU3′ or GAUCACCUCCUUUCUA3′ in B. subtilis. Such differences in 3′TAIL lead to species-specific SDs (designated SDEc for E. coli and SDBs for B. subtilis) that can form strong and well-positioned SD/aSD pairing in one species but not in the other. Selection mediated by the species-specific 3′TAIL is expected to favor SDBs against SDEc in B. subtilis, but favor SDEc against SDBs in E. coli. Among well-positioned SDs, SDEc is used more in E. coli than in B. subtilis, and SDBs more in B. subtilis than in E. coli. Highly expressed genes and genes of high translation efficiency tend to have longer SDs than lowly expressed genes and genes with low translation efficiency in both species, but more so in B. subtilis than in E. coli. Both species overuse SDs matching the bolded part of the 3′TAIL shown above. The 3′TAIL difference contributes to the host specificity of phages.

The Frequency of Internal Shine-Dalgarno-like Motifs in Prokaryotes.

In prokaryotes, translation initiation typically depends on complementary binding between a G-rich Shine–Dalgarno (SD) motif in the 5′ untranslated region of mRNAs, and the 3′ tail of the 16S ribosomal RNA (the anti-SD sequence). In some cases, internal SD-like motifs in the coding region generate “programmed” ribosomal pauses that are beneficial for protein folding or accurate targeting. On the other hand, such pauses can also reduce protein production, generating purifying selection against internal SD-like motifs. This selection should be stronger in GC-rich genomes that are more likely to harbor the G-rich SD motif. However, the nature and consequences of selection acting on internal SD-like motifs within genomes and across species remains unclear. We analyzed the frequency of SD-like hexamers in the coding regions of 284 prokaryotes (277 with known anti-SD sequences and 7 without a typical SD mechanism). After accounting for GC content, we found that internal SD-like hexamers are avoided in 230 species, including three without a typical SD mechanism. The degree of avoidance was higher in GC-rich genomes, mesophiles, and N-terminal regions of genes. In contrast, 54 species either showed no signature of avoidance or were enriched in internal SD-like motifs. C-terminal gene regions were relatively enriched in SD-like hexamers, particularly for genes in operons or those followed closely by downstream genes. Together, our results suggest that the frequency of internal SD-like hexamers is governed by multiple factors including GC content and genome organization, and further empirical work is necessary to understand the evolution and functional roles of these motifs.

Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis

Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30s−1 at 1mM free Mg2+ concentration to the detrimentally low value of 1s−1 at 6mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0–6kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account.

Translation Enhancer Improves the Ribosome Liberation from Translation Initiation

For translation initiation in bacteria, the Shine-Dalgarno (SD) and anti-SD sequence of the 30S subunit play key roles for specific interactions between ribosomes and mRNAs to determine the exact position of the translation initiation region. However, ribosomes also must dissociate from the translation initiation region to slide toward the downstream sequence during mRNA translation. Translation enhancers upstream of the SD sequences of mRNAs, which likely contribute to a direct interaction with ribosome protein S1, enhance the yields of protein biosynthesis. Nevertheless, the mechanism of the effect of translation enhancers to initiate the translation is still unknown. In this paper, we investigated the effects of the SD and enhancer sequences on the binding kinetics of the 30S ribosomal subunits to mRNAs and their translation efficiencies. mRNAs with both the SD and translation enhancers promoted the amount of protein synthesis but destabilized the interaction between the 30S subunit and mRNA by increasing the dissociation rate constant (koff) of the 30S subunit. Based on a model for kinetic parameters, a 16-fold translation efficiency could be achieved by introducing a tandem repeat of adenine sequences (A20) between the SD and translation enhancer sequences. Considering the results of this study, translation enhancers with an SD sequence regulate ribosomal liberation from translation initiation to determine the translation efficiency of the downstream coding region.

Unexpected variations in translation initiation machinery

Background In prokaryotes, the Shine-Dalgarno (SD) sequence in the 5’ untranslated region (UTR) of mRNA forms a duplex with the anti-Shine-Dalgarno (anti-SD) sequence to initiate translation [1]. The mechanism has been regarded as universal, as the core of the anti-SD sequence (5’-CCTCC3’, anti-SD motif) is completely conserved in every prokaryote genome according to previous reports [2]. Recent accelerated accumulation of complete genomic sequences motivated us to conduct an updated screening for anti-SD sequence variants.

Anti-Shine–Dalgarno regulation of translation

The authors performed an analysis of ribosome pausing in Escherichia coli and Bacillus subtilis by ribosome profiling — a technique that allows the identification of ribosome-protected mRNA by high-throughput sequencing. They show that ribosome pausing is mediated by Shine–Dalgarno (SD)-like sequences within coding regions; this pausing seems to be due to an altered 16S anti-SD sequence within the ribosome that hybridizes with the SD-like sequence. In coding sequences, SD-like sequences are disfavoured and their inclusion is a way in which translation is controlled.

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

Protein synthesis by ribosomes takes place on a linear substrate but at variable speeds. Transient pausing of ribosomes can impact a variety of co-translational processes, including protein targeting and folding1. These pauses are influenced by the sequence of the mRNA2. 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 using ribosome profiling–deep sequencing of ribosome-protected mRNA fragments3-5. This approach enables high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare tRNAs do not lead to slow translation under nutrient-rich conditions. Instead, Shine-Dalgarno-(SD)6 like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome7,8 possessing an altered anti-SD sequence, we demonstrated that pausing is due to hybridization between mRNA and the 16S rRNA 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.

Shine-Dalgarno sequence of bacteriophage T4: GAGG prevails in early genes

Translation initiation is governed by a limited number of mRNA sequence motifs within the translation initiation region (TIR). In bacteria and bacteriophages, one of the most important determinants is a Shine-Dalgarno (SD) sequence that base pairs with the anti-SD sequence GAUCACCUCCUUA localized in the 3' end of 16S rRNA. This work assesses a diversity of TIR features in phage T4, focusing on the SD sequence, its spacing to the start codon and relationship to gene expression and essentiality patterns. Analysis shows that GAGG is predominant of all core SD motifs in T4 and its related phages, particularly in early genes. Possible implication of the RegB activity is discussed.

The SAM‐responsive SMK box is a reversible riboswitch

The S(MK) (SAM-III) box is an S-adenosylmethionine (SAM)-responsive riboswitch found in the 5' untranslated region of metK genes, encoding SAM synthetase, in many members of the Lactobacillales. SAM binding causes a structural rearrangement in the RNA that sequesters the Shine-Dalgarno (SD) sequence by pairing with a complementary anti-SD (ASD) sequence; sequestration of the SD sequence inhibits binding of the 30S ribosomal subunit and prevents translation initiation. We observed a slight increase in the half-life of the metK transcript in vivo when Enterococcus faecalis cells were depleted for SAM, but no significant change in overall transcript abundance, consistent with the model that this riboswitch regulates at the level of translation initiation. The half-life of the SAM-S(MK) box RNA complex in vitro is shorter than that of the metK transcript in vivo, raising the possibility of reversible binding of SAM. We used a fluorescence assay to directly visualize reversible switching between the SAM-free and SAM-bound conformations. We propose that the S(MK) box riboswitch can make multiple SAM-dependent regulatory decisions during the lifetime of the transcript in vivo, acting as a reversible switch that allows the cell to respond rapidly to fluctuations in SAM pools by modulating expression of the SAM synthetase gene.

Dynamic evolution of translation initiation mechanisms in prokaryotes

It is generally believed that prokaryotic translation is initiated by the interaction between the Shine-Dalgarno (SD) sequence in the 5' UTR of an mRNA and the anti-SD sequence in the 3' end of a 16S ribosomal RNA. However, there are two exceptional mechanisms, which do not require the SD sequence for translation initiation: one is mediated by a ribosomal protein S1 (RPS1) and the other used leaderless mRNA that lacks its 5' UTR. To understand the evolutionary changes of the mechanisms of translation initiation, we examined how universal the SD sequence is as an effective initiator for translation among prokaryotes. We identified the SD sequence from 277 species (249 eubacteria and 28 archaebacteria). We also devised an SD index that is a proportion of SD-containing genes in which the differences of GC contents are taken into account. We found that the SD indices varied among prokaryotic species, but were similar within each phylum. Although the anti-SD sequence is conserved among species, loss of the SD sequence seems to have occurred multiple times, independently, in different phyla. For those phyla, RPS1-mediated or leaderless mRNA-used mechanisms of translation initiation are considered to be working to a greater extent. Moreover, we also found that some species, such as Cyanobacteria, may acquire new mechanisms of translation initiation. Our findings indicate that, although translation initiation is indispensable for all protein-coding genes in the genome of every species, its mechanisms have dynamically changed during evolution.

Questionable 16S ribosomal RNA gene annotations are frequent in completed microbial genomes

According to recent reports, many ribosomal RNA gene annotations are still questionable, and the use of inappropriate tools for annotation has been blamed. However, we believe that the abundant 16S rRNA partial sequence in the databases, mainly created by culture-independent PCR methods, is another main cause of the ambiguous annotations of 16S rRNA. To examine the current status of 16S rRNA gene annotations in complete microbial genomes, we used as a criterion the conserved anti-SD sequence, located at the 3′ end of the 16S rRNA gene, which is commonly overlooked by culture-independent PCR methods. In our large survey, 859 16S rRNA gene sequences from 252 different species of the microbial complete genomes were inspected. 67 species (234 genes) were detected with ambiguous annotations. The common anti-SD sequence and other conserved 16S rRNA sequence features could be detected in the downstream-intergenic regions for almost every questionable sequence, indicating that many of the 16S rRNA genes were annotated incorrectly. Furthermore, we found that more than 91.5% of the 93,716 sequences of the available 16S rRNA in the main databases are partial sequences. We also performed BLAST analysis for every questionable rRNA sequence, and most of the best hits in the analysis were rRNA partial sequences. This result indicates that partial sequences are prevalent in the databases, and that these sequences have significantly affected the accuracy of microbial genomic annotation. We suggest that the annotation of 16S rRNA genes in newly complete microbial genomes must be done in more detail, and that revision of questionable rRNA annotations should commence as soon as possible.

A Snapshot of the 30S Ribosomal Subunit Capturing mRNA via the Shine-Dalgarno Interaction

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.