Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome.

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 39 end of 16S rRNA (aSD) complementary to the ShineDalgarno (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 b-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 b-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 59 untranslated region, it can ensure a substantial enhancing effect on translation.

We have shown previously that when the Escherichia coli chromosomal lacZ gene is put under the control of an extended Shine-Dalgarno (SD) sequence (10 or 6 nucleotides in length), the translation efficiency can be highly variable, depending on the presence of AU-rich targets for ribosomal protein S1 in the mRNA leader. Here, the same strains have been used to examine the question of how strong ribosome binding to extended SD sequences affects the stability of lacZ mRNAs translated with different efficiencies. The steady-state concentration of the lacZ transcripts has been found to vary over a broad range, directly correlating with translation efficiency but not with the SD duplex stability. The observed strain-to-strain variations in lacZ mRNA level became far less marked in the presence of the rne-1 mutation, which partially inactivates RNase E. Together, the results show that (i) an SD sequence, even one that is very long, cannot stabilize the lacZ mRNA in E. coli if translation is inefficient; (ii) inefficiently translated lacZ transcripts are sensitive to RNase E; and (iii) AU-rich elements inserted upstream of a long SD sequence enhance translation and stabilize mRNA, despite the fact that they constitute potential RNase E sites. These data strongly support the idea that the lacZ mRNA in E. coli can be stabilized only by translating, and not by stalling, ribosomes.As a rule, prokaryotic mRNAs are unstable, with half-lives in the minute range. In Escherichia coli, pathways of mRNA decay are complex and include an initial rate-limiting endonucleolytic cleavage (generally mediated by RNase E) followed by 3Ј35Ј exonucleolytic degradation of the cleavage products (reviewed in references 6, 10, 19, and 34). For several mRNAs, decay pathways have been studied in detail, allowing molecular models to be formulated (4-6, 11, 13, 28). Several lines of evidence suggest that translating ribosomes usually protect the mRNA from endonucleolytic attacks (10). Much of the available information has been derived from experiments with lacZ translational fusions. In this case, mRNA translation and degradation are intimately correlated. In particular, mutations in the ribosome binding site (RBS) which reduce translation initiation efficiency, and hence efficiency of the overall translation, accelerate mRNA degradation (14,22,38). However, despite all efforts, the mechanism of the lacZ mRNA decay still remains poorly characterized.Although in many cases translation efficiency positively correlates with stability of the lacZ mRNA in E. coli, the question of whether merely the ribosome binding to the RBS is able to stabilize the whole transcript remains equivocal. On one hand, it has been suggested that a ribosome bound to a strong ShineDalgarno (SD) sequence, but not translation per se, could be the main stability factor for the lacZ mRNA (37). This would imply that E. coli mRNAs can be protected from degradation by the same mechanism as in bacilli, where ribosome stalling within 5Ј untranslated regions can protect kilobases of the...

The ssyF29 mutation, originally selected as an extragenic suppressor of a protein export defect, has been mapped within the rpsA gene encoding ribosomal protein S1. Here, we examine the nature of this mutation and its effect on translation. Sequencing of the rpsA gene from the ssyF mutant has revealed that, due to an IS10R insertion, its product lacks the last 92 residues of the wild-type S1 protein corresponding to one of the four homologous repeats of the RNA-binding domain. To investigate how this truncation affects translation, we have created two series of Escherichia coli strains (rpsA ؉ and ssyF) bearing various translation initiation regions (TIRs) fused to the chromosomal lacZ gene. Using a ␤-galactosidase assay, we show that none of these TIRs differ in activity between ssyF and rpsA ؉ cells, except for the rpsA TIR: the latter is stimulated threefold in ssyF cells, provided it retains at least ca. 90 nucleotides upstream of the start codon. Similarly, the activity of this TIR can be severely repressed in trans by excess S1, again provided it retains the same minimal upstream sequence. Thus, the ssyF stimulation requires the presence of the rpsA translational autogenous operator. As an interpretation, we propose that the ssyF mutation relieves the residual repression caused by normal supply of S1 (i.e., that it impairs autogenous control). Thus, the C-terminal repeat of the S1 RNA-binding domain appears to be required for autoregulation, but not for overall mRNA recognition.

No abstract

In an attempt to understand how Escherichia coli ribosomes recognize the initiator codon on mRNAs lacking the Shine-Dalgarno (SD) sequence, we have studied 30s initiation complex formation in extension inhibition (toeprinting) experiments using (-SD)mRNAs which are known to be reliably translated in E. coli: the plant viral messenger AlMV RNA 4 and two chimaeric mRNAs coding for p-glucuronidase (GUS) and bearing the S-untranslated sequence of TMV RNA (Q) or the O-derived sequence (CAA), as S-leaders. Ribosomal protein Sl and IF3 have been found to be indispensable for translational initiation. Protein Sl appears to be a key recognition element. Sl binds to sequences within the leaders of (-SD)mRNAs thus providing their affinity to E. coli ribosomes.

High-affinity RNA ligands were generated against intact 30S ribosomes, S1-depleted 30S ribosomes, and purified ribosomal protein S1. Sequence analysis indicated two classes of ligand: unstructured RNAs containing a Shine-Dalgarno sequence and structured RNAs containing a pseudoknot. The Shine-Dalgarno-containing ligands were generated against S1-depleted 30S ribosomes but, surprisingly, not against intact 30S ribosomes or ribosomal protein S1. In contrast, pseudoknot-containing ligands were generated against intact ribosomes as well as purified S1 protein. The two classes of ligand exhibited specificity for their respective targets, as well as conserved sequence and secondary structure reminiscent of naturally occurring, cis-acting mRNA elements.

Translation initiation region (TIR) of the rpsA mRNA encoding ribosomal protein S1 is one of the most ef®-cient in Escherichia coli despite the absence of a canonical Shine±Dalgarno-element. Its high ef®ciency is under strong negative autogenous control, a puzzling phenomenon as S1 has no strict sequence speci®-city. To de®ne sequence and structural elements responsible for translational ef®ciency and autoregulation of the rpsA mRNA, a series of rpsA¢±¢lacZ chromosomal fusions bearing various mutations in the rpsA TIR was created and tested for b-galactosidase activity in the absence and presence of excess S1. These in vivo results, as well as data obtained by in vitro techniques and phylogenetic comparison, allow us to propose a model for the structural and functional organization of the rpsA TIR speci®c for proteobacteria related to E.coli. According to the model, the high ef®ciency of translation initiation is provided by a speci®c fold of the rpsA leader forming a non-contiguous ribosome entry site, which is destroyed upon binding of free S1 when it acts as an autogenous repressor.