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.     

Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation

There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving beta-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the beta-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5' untranslated region, it can ensure a substantial enhancing effect on translation.     

Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences.

We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.     

Mutagenesis at the mRNA decoding site in the 16S ribosomal RNA using the specialized ribosome system in Escherichia coli.

In the specialized ribosome system, a distinct pool of mutated ribosomes is dedicated to the translation of one particular mRNA species. This was accomplished by altering the Shine-Dalgarno sequence on the mRNA and its complementary anti-Shine-Dalgarno sequence on the plasmid-borne 16S rRNA gene. Here, using the specialized ribosome system, we were able to introduce mutations in key regions of the 16S rRNA and could study their effect on translation in vivo. The C1400 region has been implicated to play a role in the actual mRNA decoding process. Several ribosomal mutations were introduced in this region. We showed that substitution of the evolutionary highly conserved C1400 residue by a G- or an A-residue inhibits ribosomal activity by 80% and 50% respectively, whereas, a C to a U change at this conserved position does not affect overall ribosomal activity. The adjacent stem structure (1410-1490) was also examined. Disruption of the stem by replacing either one of the arms of this stem, with a different sequence, inhibits ribosomal activity by approximately 80%. A small but significant restoration of translation could be achieved by recreating a complementary stem with a different sequence. We found that full reversion of activity could be obtained when such mutated ribosomes were made spectinomycin resistant by introducing a C to A substitution at position 1192 which is located far away in the secondary structure map of the 16S rRNA molecule. Based on these results we conclude that some, but not all, of the nucleotides in the conserved C1400 region play a key role in translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

An antisense/target RNA duplex or a strong intramolecular RNA structure 5' of a translation initiation signal blocks ribosome binding: the case of plasmid R1.

Antisense RNAs in prokaryotic systems often inhibit translation of mRNAs. In some cases, this involves sequestration of Shine-Dalgarno (SD) sequences and start codons. In other cases, antisense/target RNA duplexes do not overlap these signals, but form upstream. We have performed toeprinting analyses on repA mRNA of plasmid R1, both free and in duplex with the antisense RNA, CopA. An intermolecular RNA duplex 2 nt upstream of the tap SD prevents ribosome binding. An intrastrand stem-loop at this location yields the same inhibition. Thus, stable secondary structures immediately upstream of the tap SD sequence inhibit translation, as shown by toeprinting in vitro and repA-lacZ expression in vivo. Previous work showed that repA (initiator protein) expression requires tap (leader peptide) translation. Toeprinting data confirm that the tap ribosome binding site (RBS) is accessible, whereas the repA RBS, which is sequestered by a stable stem-loop, is weakly recognized by the ribosome. Truncated CopA RNA (CopI) is unable to pair completely with target RNA, but proceeds normally to a kissing intermediate. This mutant RNA species inhibits repA expression in vivo. By a kinetic toeprint inhibition protocol, we have shown that the structure of the kissing complex is sufficient to sterically prevent ribosome binding. These results are discussed in comparison with the effect of RNA structures elsewhere in the ribosome-binding region of an mRNA.     

Translation inhibition from a distance: The small RNA SgrS silences a ribosomal protein S1-dependent enhancer

Many bacterial small RNAs (sRNAs) efficiently inhibit translation of target mRNAs by forming a duplex that sequesters the Shine-Dalgarno (SD) sequence or start codon and prevents formation of the translation initiation complex. There are a growing number of examples of sRNA–mRNA binding interactions distant from the SD region, but how these mediate translational regulation remains unclear. Our previous work in Escherichia coli and Salmonella identified a mechanism of translational repression of manY mRNA by the sRNA SgrS through a binding interaction upstream of the manY SD. Here, we report that SgrS forms a duplex with a uridine-rich translation-enhancing element in the manY 5ʹ untranslated region. Notably, we show that the enhancer is ribosome-dependent and that the small ribosomal subunit protein S1 interacts with the enhancer to promote translation of manY. In collaboration with the chaperone protein Hfq, SgrS interferes with the interaction between the translation enhancer and ribosomal protein S1 to repress translation of manY mRNA. Since bacterial translation is often modulated by enhancer-like elements upstream of the SD, sRNA-mediated enhancer silencing could be a common mode of gene regulation.     

In vivo studies on putative Shine-Dalgarno sequences of the halophilic archaeon Halobacterium salinarum

The involvement of Shine-Dalgarno sequences in the translation of mRNA in halophilic archaea was investigated for two gvp genes involved in gas vesicle formation in Halobacterium salinarum PHH1. With the exception of gvpA and gvpO, all reading frames of the p-gvpDEFGHIJKLM and p-gvpACNO mRNAs contained upstream of the AUG start codon a putative Shine-Dalgarno (SD) sequence that is complementary to the 3'-end of the small ribosomal subunit RNA. The importance of the SD sequences of gvpG and gvpH was investigated in Haloferax volcanii transformants, and an alteration of the SD sequence resulted in a reduction of the amount of the GvpG or GvpH protein. For a more quantitative analysis the region upstream of gvpH was fused to the bgaH reading frame encoding an enzyme with beta-galactosidase activity as reporter. Scanning mutagenesis within the mRNA leader demonstrated that mutations adjacent to the putative SD sequence GGAGGUCA did not influence the efficiency of translation, whereas constructs harbouring an altered SD sequence yielded only 5-50% of the beta-galactosidase activities obtained with the wild-type SD element. A complete mutation of the SD sequence still yielded 20% of the wild-type activity. Alterations in the spacing of the SD sequence and the translation initiation codon of gvpH indicated that a distance of 4 or 10 nucleotides yielded a similar beta-galactosidase activity as found with the 7 nt spacing of the SD element in wild type, whereas a distance of 1 nt resulted in the loss of translation. A complete deletion of the 5'-UTR resulting in a leaderless mRNA yielded an enhanced beta-galactosidase activity in transformants implying that the initiation of translation involved a mechanism other than a specific mRNA-rRNA interaction.     

Cis-acting elements and trans-acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts.

Translational regulation is an important step of gene expression in chloroplasts. To analyze biochemical mechanisms of translational regulation unique to higher plant chloroplasts, an in vitro translation system has been developed from tobacco chloroplasts. Conditions for chloroplast extraction and the in vitro translation reaction have been optimized with a tobacco psbA-lacZ fusion mRNA. The in vitro system supports accurate translation of a variety of chloroplasts mRNAs. Using a series of mutant psbA mRNAs, we showed that three elements within the 5'-untranslated region of the mRNA are required for translation. Two of them are complementary to the 3'-terminus of chloroplast 16S rRNA (termed RBS1 and RBS2) and the other is an AU-rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed the AU box. mRNA competition experiments using the in vitro translation reaction and gel mobility shift assays revealed the existence of a trans-acting factor(s) for translation and its possible interaction with the AU box. We propose a model for the initiation of psbA translation whereby RBS1 and RBS2 bind cooperatively to the 3'-end of 16S rRNA resulting in looping out of the AU box, which facilitates the interaction of a trans-acting factor(s).     

mRNA containing an extended Shine-Dalgarno sequence is translated independently of ribosomal protein S1.

The role of ribosomal protein S1 in the translation of mRNA containing an extended Shine-Dalgarno sequence was investigated. Using the toeprinting technique, formation of the ternary initiation complex between 30S subunits, both S1-depleted or treated with anti-S1 antibodies, and mini-mRNA containing the 9 nucleotide-long Shine-Dalgarno sequence was studied. It was concluded that the initiation of translation on mRNA with an extended Shine-Dalgarno sequence is S1-independent. It was demonstrated that S1-depleted ribosomes effectively translate the cro-mini-mRNA in a cell-free system. In contrast to cro-mini-mRNA, 30S subunits without protein S1 are inactive in ternary initiation complex formation with, and cell-free translation of, MS2 or fr phage RNAs and RNA protein III of phage fd.     

Identification and characterization of E.coli ribosomal binding sites by free energy computation.

Sequences upstream from translational initiation sites of different E.coli genes show various degrees of complementarity to the Shine-Dalgarno (SD) sequence at the 3' end of the 16S rRNA. We propose a quantitative measure for the SD region on the mRNA, that reflects its degree of complementarity to the rRNA. This measure is based on the stability of the rRNA-mRNA duplex as established by free energy computations. The free energy calculations are based on the same principles that are used for folding a single RNA molecule, and are executed by similar algorithms. Bulges and internal loops in the rRNA and mRNA are allowed. The mRNA string with maximum free energy gain upon binding to the rRNA is selected as the most favorable SD sequence of a gene. The free energy value that represents the SD region provides a quantitative measure that can be used for comparing SD sequences of different genes. The distribution of this measure in more than 1000 E.coli genes is presented and discussed.     

The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4-6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 microM copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.     

The initiation of translation in E. coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA.

Bacteriophage T7's gene 0.3, coding for an antirestriction protein, possesses one of the strongest translation initiation regions (TIR) in E. coli. It was isolated on DNA fragments of differing length and cloned upstream of the mouse dihydrofolate reductase gene in an expression vector to control the translation of this gene's sequence. The TIR's efficiency was highly dependent on nucleotides +15 to +26 downstream of the gene's AUG. This sequence is complementary to nucleotides 1471-1482 of the 16srRNA. Similar sequences complementary to this rRNA region are present in other efficient TIRs of the E. coli genome and those of its bacteriophages. There seems to be a correlation between this sequence homology and the efficiency of the initiation signals. We propose that this region specifies a stimulatory interaction between the mRNA and 16srRNA besides the Shine-Dalgarno interaction during the translation initiation step.     

An adenosyl-cobalamin (coenzyme-B12)-repressed translational enhancer in the cob mRNA of Salmonella typhimurium

Expression of the cobalamin (Cbl) biosynthetic cob operon in Salmonella typhimurium is repressed by the end-product. This regulation is conferred mainly at the translational level and involves a cobalamin-induced folding of an RNA hairpin that sequesters the ribosomal binding site (RBS) of the cob mRNA and prevents translation initiation. A combined structural and mutational analysis shows that a cis-acting translational enhancer (TE) element, located 83 nucleotides upstream of the Shine-Dalgarno sequence in the 5'-untranslated region (5'-UTR) of the cob mRNA, is required to unfold the inhibitory RBS hairpin in the absence of cobalamin. The TE element, which consists of 5 nucleotides, is proposed to confer its enhancer function in the absence of cobalamin by interacting with nucleotides in the stem of the RBS hairpin. This interaction destabilizes the RNA hairpin and allows ribosome binding. In the presence of cobalamin, the enhancer function is inhibited. As a result, the RBS hairpin forms and prevents translation initiation. Several additional RNA hairpins in the 5'-UTR were also identified and are suggested to be important for repression. The above data suggest that normal cobalamin repression of the cob operon requires that the 5'-UTR has a defined secondary and tertiary structure.     

Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation. 2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human gamma-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied. 3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell. 4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.     

Translational enhancement by an element downstream of the initiation codon in Escherichia coli

The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence. This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs. It has been debated that a cis element called the downstream box downstream of the initiation codon, in addition to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive. Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD.     

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.     

Inability of Prevotella bryantii to form a functional Shine-Dalgarno interaction reflects unique evolution of ribosome binding sites in Bacteroidetes

The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5' untranslated mRNA regions (5'-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5'-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5'-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5'-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished.     

Only one out of the three strong ribosomal binding sites of the early region of bacteriophage T7 exhibits high translational efficiency in fragments of about 30 base pairs

Within the early region of bacteriophage T7 three genes, 0.3, 1 and 1.3, are most efficiently expressed. They belong to the strongest initiation signals of Escherichia coli. In the T7 wild-type situation the proteins are produced with a molar ratio of gene 1:1.3:0.3 protein = 1:3.9:9.7. DNA fragments of about 30 base pairs comprising the ribosomal binding sites (RBS) of these genes were synthesized and cloned into derivatives of the pDS1 vector ribosomal binding sites (RBS) of these genes were synthesized and cloned into two derivatives of the pDS1 vector just upstream of the mouse dihydrofolate reductase gene. Although all tested RBS fragments contained an initiation triplet, a Shine-Dalgarno sequence and some nucleotides upstream and downstream of this region, only the gene 1.3 RBS fragment showed high efficiency whereas those of genes 0.3 and 1 were at the border of significance. The amount of synthesized mRNA was about the same for all three constructs. A major influence of vector-derived sequences on the RBS activity could be ruled out. The high translational activity of the short 1.3 gene RBS seems to be largely due to its primary structure. The other two RBSs studied require much longer sequences for high activity.