Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs.

The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon. It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene. Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3'-end of the 16S rRNA and the P-site, we conclude that there is a unique optimal aligned SD-AUG spacing in the absence of other complicating issues.     

The regulatory region of MS2 phage RNA replicase cistron. IV. Functional activity of specific MS2 RNA fragments in formation of the 70 S initiation complex of protein biosynthesis.

The initiation region of the MS2 replicase cistron can be isolated as a fragment 59 bases in length protected from RNAase by the binding of the coat protein which serves as a translational repressor. This fragment MS2 R(-53 leads to 6) starts 53 bases before the initiation codon and retains full activity in binding ribosomes. We have investigated the functional activity in initiation of a series of fragments from this region variously shortened from the 5'-end. Ribosome protected fragments starting 17 or 21 bases before the AUG are unable to rebind to ribosomes. The shortest fragment which has this activity was produced by partial S1 nuclease digestion and starts 33 to 35 bases before the AUG. The initiation signal comprises some nucleotides between 21 and 33 bases before the initiation codon and the regulatory region responsible for initiation is longer than that protected by the ribosome in the final initiation complex.     

The downstream box: an efficient and independent translation initiation signal in Escherichia coli.

The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.     

The Escherichia coli threonyl-tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation

The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase (ThrRS) is negatively autoregulated at the translational level. ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine–Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single-stranded region (domain 3). Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non-contiguous sites: region −12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single-stranded sequence in domain 3. These two regions are brought into close proximity by a 38-nucleotide-long hairpin structure (domain 2). This domain, although adjacent to the 5' edge of the SD sequence, does not inhibit ribosome binding as long as the single-stranded region of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation. As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance.     

Non-canonical RNA arrangement in T4-even phages: accommodated ribosome binding site at the gene 26-25 intercistronic junction

Translational initiation region of bacteriophage T4 gene 25 contains three potential Shine and Dalgarno sequences: SD1, SD2 and SD3. Mutational analysis has predicted that an mRNA stem-loop structure may include SD1 and SD2, bringing the most typical sequence SD3, GAGG, to the initiation codon. Here, we report physical evidence demonstrating that previously predicted mRNA stem-loop structure indeed exists in vivo during gene 25 expression in T4-infected Escherichia coli cells. The second mRNA stem-loop structure is identified 14 nucleotides upstream of the stem-loop I, while the SD3 sequence, as well as the start codon of the gene, are proved to be within an unfolded stretch of mRNA. Phylogenetic comparison of 38 T4-like phages reveals that the T-even and some pseudoT-even phages evolve a similar structural strategy for the translation initiation of 25 , while pseudoT-even, schizoT-even and exoT-even phages use an alternative mRNA arrangement. Taken together, the results indicate that a specific mRNA fold forms the split ribosome binding site at the gene 26-25 intercistronic junction, which is highly competent in the translational initiation. We conclude that this ribosome binding site has evolved after T-even diverged from other T4-like phages. Additionally, we determine that the SD sequence GAGG is most widespread in T4.     

Analysis of elements involved in pseudoknot-dependent expression and regulation of the repA gene of an IncL/M plasmid

Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation of repB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of repA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.     

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.     

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.     

Quantitative correlation between mRNA secondary structure around the region downstream of the initiation codon and translational efficiency in Escherichia coli

Translational efficiency in Escherichia coli is known to be strongly influenced by the secondary structure around the ribosome‐binding site and the initiation codon in the translational‐initiation region of the mRNA. Several quantitative studies have reported that translational efficiency is attributable to effects on ribosome accessibility predominantly caused by the secondary structure surrounding the ribosome‐binding site. However, the influence of mRNA secondary structure around regions downstream of the initiation codon on translational efficiency after ribosome‐binding step has not been quantitatively studied. Here, we quantitatively analyzed the relationship between secondary structure of mRNA surrounding the region downstream of the initiation codon, referred to as the downstream region (DR), and protein expression levels. Modified hairpin structures containing the initiation codon were constructed by site‐directed mutagenesis, and their effects on expression were analyzed in vivo. The minimal folding free energy (ΔG) of a local hairpin structure was found to be linearly correlated with the relative expression level over a range of fourfold change. These results demonstrate that expression level can be quantitatively controlled by changing the stability of the secondary structure surrounding the DR. Biotechnol. Bioeng. 2009; 104: 611–616 © 2009 Wiley Periodicals, Inc.     

The influence of mRNA secondary structure on expression of endoglucanase inBacillus subtilis

Summary A gene for endoglucanase ofBacillus subtilis has been inserted into a Bacillus expression plasmid containing a strong BJ27 promoter and a synthetic ribosome binding site. Secondary structure analysis of mRNA showed the presence of a strong hairpin loop burying the SD sequence and the initiation codon. Alteration of secondary structure at this site by deletion analysis revealed a correlation between endoglucanase expression and accessibility of the ribosome binding site. Elimination of secondary structures increased endoglucanase expression over five-fold to a level at which endoglucanase occupied 60% of total protein which was secreted into culture medium.     

Essential elements in the coding region of mRNA for translation of ColE2 Rep protein

Translation initiation of mRNA encoding the plasmid-specified initiator protein (Rep) required for initiation of the ColE2 plasmid DNA replication is fairly efficient in Escherichia coli despite the absence of a canonical Shine-Dalgarno sequence. Although a GA cluster sequence exists upstream the initiation codon, its activity as the SD sequence has been shown to be very inefficient. Deletion analyses have shown that there are sequences important for the Rep translation in the regions upstream the GA cluster sequence and downstream the initiation codon. To further define regions important for translation of the Rep mRNA, a set of the ColE2 rep genes bearing single-base substitution mutations in the coding region near the initiation codon was generated and their translation activities examined. We showed that translation of the Rep mRNA was reduced by some of these mutations in a region ranging at least 70 nucleotides from the initiation codon in the coding region, indicating the presence of translation enhancer(s) outside the translation initiation region which is covered by the ribosome bound to the initiation codon. Some of them seem to be essential and specific for translation of the ColE2 Rep mRNA due to the absence of a canonical SD sequence.     

In vitro, the major ribosome binding site on Rous sarcoma virus RNA does not contain the nucleotide sequence coding for the N-terminal amino acids of the gag gene product.

Ribosomes from the reticulocyte lysate bind strongly and mainly to a region located in the 5' end of the Rous sarcoma virus RNA molecule between residues 9 and 53. This binding involves the participation of initiator tRNA and is sensitive to inhibitors of initiation of protein synthesis such as 7-methyl-GMP and aurintricarboxylic acid. The nucleotide sequence of this ribosome binding site has been determined: it conatains a GUG codon centered at position 26 that is not in phase with any termination codon within the 5' end nucleotide sequence of the RNA that we have analyzed (101 residues). However, the predicted N-terminal amino acid sequence starting from this GUG codon (or even from any AUG or GUG codon in the 5' end of the RNA) does not coincide with that of the in vitro-synthesized product of the 5' end proximal gag gene. Nevertheless, inhibition of ribosome binding to this site is accompanied by an inhibition of the in vitro translation of the gag gene.     

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.     

A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.     

Cooperative effects by the initiation codon and its flanking regions on translation initiation

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.     

Nucleotide sequence of the Clostridium thermocellum laminarinase gene.

The sequence presented (1022 bp) shows the Clostridium thermocellum laminarinase gene (lam1) and its flanking regions. The gene lam1 comprises an open reading frame of 726 nt, encoding a 242-aa protein with predicted Mr 27661. The ORF startswith the translation initiation codon ATG. This ATG codon is preceded at a spacing of 7 bp by a potential ribosome binding site (GGAGGT). A putative signal peptide was identified (the potential cleavage site is between position 27-28 aa). The comparison of the primary protein sequence with other beta-1, 3-1, 4-glucanases showed extensive homology for Bacillus amyloliqefaciens and Bacillus subtilis glucanases (identity-46.7%; similarity-57.0%).