Two different mechanisms for ribosome/mRNA interaction in archaeal translation initiation

In this study, we have analysed the features of mRNA/ribosome interaction in the thermophilic archeon Sulfolobus solfataricus. Leadered mRNAs endowed with ShineDalgarno (SD) motifs formed stable binary complexes with 30S subunits, optimally at high temperature (6570 degrees C) and without the aid of initiator tRNA (tRNAi) or any factor. 'Toeprinting' assays revealed that the SD motifs were necessary and sufficient to direct the 30S subunit to the translation initiation region. Leaderless mRNAs, i.e. mRNAs entirely lacking a 5'-untranslated region (UTR), did not interact directly with 30S subunits but required the presence of tRNAi, indicating that codonanticodon pairing was required for positioning the ribosome on the initiation codon. The data suggest that archaea such as Sulfolobus routinely use two distinct mechanisms for translational initiation. SD-dependent initiation, resembling the pathway prevalent in present-day bacteria, would operate on distal cistrons of polycistronic mRNAs, whereas 'leaderless' initiation, reminiscent of the eukaryotic pathway, would operate on monocistronic mRNAs and on opening cistrons of polycistronic mRNAs.     

Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation.

Secondary structure of the mRNA in the translational initiation region is an important determinant of translation efficiency. However, the secondary structures that enhance or facilitate translation initiation are rare. We have previously proposed that such structure may exist in the case of bacteriophage T4 gene 25 translational initiation region, which contains three potential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17, and 27 nucleotides from the initiation codon of this gene, respectively. We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25.Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a decreased level of gp25 synthesis. Double mutation in base-pair V restores the level of gene 25 expression that was decreased by either of the two mutations (C-to-G and G-to-C) alone, as predicted by an effect attributable to mRNA secondary structure. We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination. Changes in the plaque and burst sizes of T4 mutants, carrying single and double mutations in the translational initiation region of gene 25, strongly suggest that the predicted mRNA secondary structure controls (enhances) the level of gene 25 expression in vivo. Hybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation region do not notably affect the 25 mRNA stability. Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold. Since the secondary structure increases the level of gp25 synthesis and does not affect mRNA stability, we conclude that this structure enhances translation initiation. We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38.     

Destabilization of messenger RNA/complementary DNA duplexes by the elongating 80 S ribosome

In a previous study, we demonstrated that the ability of a cDNA fragment to hybrid-arrest the translation of its complementary mRNA in rabbit reticulocyte lysate depends on the position of the mRNA/cDNA duplex within the mRNA molecule. In the present report, we further characterize the mechanisms involved in the destabilization and subsequent translation of mRNA/cDNA hybrids by mapping in detail the positional dependence of hybrid-arrested translation of the human alpha- and beta-globin mRNAs and by directly assessing the stability of mRNA/cDNA duplexes in reticulocyte lysate under a variety of translational conditions. The mapping studies in this report demonstrate that the translation of a hybridized mRNA requires exposure of the 5' nontranslated region and the AUG initiation codon, as well as those bases 3' to the AUG which are typically protected by an initiating 80 S ribosome. The translation of these mRNA/cDNA hybrids is associated with the complete removal of cDNA from the mRNA coding region; this disruption of the mRNA/cDNA duplex is blocked by inhibitors of translational initiation and elongation. cDNAs which extend into the 3' nontranslated region remain associated with the mRNA during normal translation but are completely removed from the mRNA during translation if translational termination is suppressed. Taken together, these findings demonstrate that the disruption of mRNA/cDNA duplexes in rabbit reticulocyte lysate is tightly linked to the assembly and migration of 80 S ribosomes.     

Mutations affecting pseudoknot control of the replication of B group plasmids.

The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure. Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major effects on the translation of repA.     

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

We determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5'-AGGA-3' to 5'-UUUU-3' results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon–anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.     

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.     

Translation initiation region sequence preferences in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation.     

Temperature-dependent translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with a 5'-terminal start codon occur in all biological systems. In this work, we have studied the comparative translational efficiency of leaderless and leadered mRNAs as a function of temperature by in vitro translation competition assays with Escherichia coli extracts. At low temperature (25 degrees C) leaderless mRNAs were found to be translated comparatively better than mRNAs containing an internal canonical ribosome binding site, whereas at high temperature (42 degrees C) the translational efficiency of canonical mRNAs is by far superior to that of leaderless mRNA. The inverse correlation between temperature and translational efficiency characteristic for the two mRNA classes was attributed to structural features of the mRNA(s) and to the reduced stability of the translation initiation complex formed at a 5'-terminal start codon at elevated temperature.     

Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control

It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5'-terminal AUG that seems to be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome-IF2-fMet-tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of leaderless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evolutionarily conserved function of the translational apparatus.     

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.     

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.     

Modulation of ribosomal recruitment to 5'-terminal start codons by translation initiation factors IF2 and IF3

Sequence determinants and structural features of the RNA govern mRNA-ribosome interaction in bacteria. However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine-Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA-mRNA interactions. Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5'-terminal AUG and at a competing downstream canonical ribosome binding site. We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5'-terminal AUG as well as the stability of these complexes. Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery.     

The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.     

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.     

A Saccharomyces cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity.

The TIF3 gene of Saccharomyces cerevisiae was cloned and sequenced. The deduced amino acid sequence shows 26% identity with the sequence of mammalian translation initiation factor eIF-4B. The TIF3 gene is not essential for growth; however, its disruption results in a slow growth and cold-sensitive phenotype. In vitro translation of total yeast RNA in an extract from a TIF3 gene-disrupted strain is reduced compared with a wild-type extract. The translational defect is more pronounced at lower temperatures and can be corrected by the addition of wild-type extract or mammalian eIF-4B, but not by addition of mutant extract. In vivo translation of beta-galactosidase reporter mRNA with varying degree of RNA secondary structure in the 5' leader region in a TIF3 gene-disrupted strain shows preferential inhibition of translation of mRNA with more stable secondary structure. This indicates that Tif3 protein is an RNA helicase or contributes to RNA helicase activity in vivo.     

Regulation of the Escherichia coli secA Gene Is Mediated by Two Distinct RNA Structural Conformations

Expression from the secA gene, encoding a key component of the general secretory pathway of Escherichia coli, is influenced by the secretion status of the cell, autogenous translational repression, and translational coupling to the upstream gene, X. SecA binds to its mRNA in a region overlapping its ribosome binding site, thus competing with ribosomes that would initiate secA translation. Mapping of the geneX-secA mRNA secondary structure has demonstrated that the RNA can adopt two distinct conformations in solution. The first conformation arises from the base-pairing of the secA Shine-Dalgarno (SD) sequence with the geneX terminus. The second conformation, in which the secA SD sequence is no longer paired with the geneX terminus, contains a GC-rich stem upstream of the secA SD sequence. The presence of this GC-rich stem is supported by structure mapping of a mutant RNA containing a deletion in the geneX terminus. The former structure appears to be involved in translational coupling by directly linking the geneX and secA sequences, where geneX translation activates secA translational initiation through the unpairing and unmasking of the secA SD sequence. As indicated by SecA-RNA binding assays, the latter structure is probably involved in SecA binding and translational repression of the secA gene. The stabilizing effect of magnesium ions toward occlusion of the secA SD sequence supports the presence of RNA tertiary structure in this regulatory domain. Received: 30 July 1998 / Accepted: 18 September 1998     

Escherichia coli mRNAs with strong Shine/Dalgarno sequences also contain 5' end sequences complementary to domain # 17 on the 16S ribosomal RNA

A well-established feature of the translation initiation region, which attracts the ribosomes to the prokaryotic mRNAs, is a purine rich area called Shine/Dalgarno sequence (SD). There are examples of various other sequences, which despite having no similarity to an SD sequence are capable of enhancing and/or initiating translation. The mechanisms by which these sequences affect translation remain unclear, but a base pairing between mRNA and 16S ribosomal RNA (rRNA) is proposed to be the likely mechanism. In this study, using a computational approach, we identified a non-SD signal found specifically in the translation initiation regions of Escherichia coli mRNAs, which contain super strong SD sequences. Nine of the 11 E. coli translation initiation regions, which were previously identified for having super strong SD sequences, also contained six or more nucleotides complementary to box-17 on the 16S rRNA (nucleotides 418-554). Mutational analyses of those initiation sequences indicated that when complementarity to box-17 was eliminated, the efficiency of the examined sequences to mediate the translation of chloramphenicol acetyltransferase (CAT) mRNA was reduced. The results suggest that mRNA sequences with complementarity to box-17 of 16S rRNA may function as enhancers for translation in E. coli.     

Translational regulation by mRNA/protein interactions in eukaryotic cells: Ferritin and beyond

The expression of certain eukaryotic genes is – at least in part – controlled at the level of mRNA translation. The step of translational initiation represents the primary target for regulation. The regulation of the intracellular iron storage protein ferritin in response to iron levels provides a good example of translational control by a reversible RNA/protein interaction in the 5' untranslated region of an mRNA. We consider mechanisms by which mRNA/protein interactions may impede translation initiation and discuss recent data suggesting that the ferritin example may represent the ‘tip of the iceberg’ of a more general theme for translational control.