Formation of the central pseudoknot in 16S rRNA is essential for initiation of translation.

The postulated central pseudoknot formed by regions 9-13/21-25 and 17-19/916-918 of 16S rRNA of Escherichia coli is phylogenetically conserved in prokaryotic as well eukaryotic species. This pseudoknot is located at the center of the secondary structure of the 16S rRNA and connects the three major domains of this molecule. We have introduced mutations into this pseudoknot by changing the base-paired residues C18 and G917, and the effect of such mutations on the ribosomal activity was studied in vivo, using a 'specialized' ribosome system. As compared with ribosomes having the wild-type pseudoknot, the translational activity of ribosomes containing an A, G or U residue at position 18 was dramatically reduced, while the activity of mutant ribosomes having complementary bases at positions 18 and 917 was at the wild-type level. The reduced translational activity of those mutants that are incapable of forming a pseudoknot was caused by their inability to form 70S ribosomal complexes. These results demonstrate that the potential formation of a central pseudoknot in 16S rRNA with any base-paired residues at positions 18 and 917 is essential to complete the initiation process.     

Deletion of a conserved, central ribosomal intersubunit RNA bridge

Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.     

Possible interaction sites of mRNA, tRNA, translation factors and the nascent peptide in 5S, 5.8S and 28S rRNA in in vivo assembled eukaryotic ribosomal complexes.

We have investigated possible interaction sites for mRNA, tRNA, translation factors and the nascent peptide on 5S, 5.8S and 28S rRNA in in vivo assembled translational active mouse ribosomes by comparing the chemical footprinting patterns derived from native polysomes, salt-washed polysomes (mainly lacking translational factors) and salt-washed runoff ribosomes (lacking mRNA, tRNA and translational factors). Several ligand-induced footprints were observed in 28S rRNA while no reactivity changes were seen in 5S and 5.8S rRNA. Footprints derived from mRNA, tRNA and/or the nascent peptide chain were observed in domain I of 28S rRNA (hairpin 23), in domain II (helix 37/38 and helices 42 and 43 and in the eukaryotic expansion segment 15), in domain IV (helices 67 and 74) and in domain V (helices 94 and 96 and in the peptidyl transferase ring). Some of the protected sites were homologous to sites previously suggested to be involved in mRNA, tRNA and/or peptide binding in in vitro assembled prokaryotic complexes. Additional footprints were located in regions that have not previously been found involved in ligand binding. Part of these sites could derive from the nascent peptide in the exit channel of the ribosome.     

Role of protein L25 and its contact with protein L16 in maintaining the active state of <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomes <Emphasis Type="Italic">in vivo</Emphasis>

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.     

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.     

16S rRNA mutations that confer tetracycline resistance in Helicobacter pylori decrease drug binding in Escherichia coli ribosomes

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.     

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.     

Structural elements of rps0 mRNA involved in the modulation of translational initiation and regulation of E. coli ribosomal protein S15.

Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.     

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.     

The highly efficient translation initiation region from the Escherichia coli rpsA gene lacks a shine-dalgarno element

The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position -91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the "specialized ribosome" approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.     

The involvement of base 1054 in 16S rRNA for UGA stop codon dependent translational termination.

The deletion of the highly conserved cytidine nucleotide at position 1054 in E. coli 16S rRNA has been characterized to confer an UGA stop codon specific suppression activity which suggested a functional participation of small subunit rRNA in translational termination. Based on this structure-function correlation we constructed the three point mutations at site 1054, changing the wild-type C residue to an A, G or U base. The mutations were expressed from a complete plasmid encoded rRNA operon (rrnB) using a conditional expression system with the lambda PL-promoter. All three altered 16S rRNA molecules were expressed and incorporated into 70S ribosomal particles. Structural analysis of the protein and 16S rRNA moieties of the mutant ribosomes showed no differences when compared to wild-type particles. The phenotypic analysis revealed that only the 1054G base change led to a significantly reduced generation time of transformed cells, which could be correlated with the inability of the mutant ribosomes to specifically stop at UGA stop codons in vivo. The response towards UAA and UAG termination codons was not altered. Furthermore, in vitro RF-2 termination factor binding experiments indicated that the association behaviour of mutant ribosomes was not changed, enforcing the view that the UGA stop codon suppression is a direct consequence of the rRNA mutation. Taken together, these results argue for a direct participation of that 16S rRNA motif in UGA dependent translational termination and furthermore, suggest that termination factor binding and stop codon recognition are two separate steps of the termination event.     

Feedback regulation of rRNA synthesis. A mutational alteration in the anti-Shine-Dalgarno region of the 16 S rRNA gene abolishes regulation

It was shown that induction of rRNA (and ribosome) synthesis from the lambda PL promoter/operator by temperature shift-up causes a repression of rRNA and tRNA synthesis from chromosomal genes. We have carried out experiments using a similar conditional rRNA gene expression system in which a mutational alteration was introduced in the anti-Shine-Dalgarno region at the 3'-end of the 16 S rRNA gene. It was found that the repression observed with the wild-type gene was largely abolished by the mutation. It appears that ribosomes inefficient in translational initiation are unable to cause feedback regulation of rRNA synthesis. It is suggested that the cell regulates rRNA (and tRNA) synthesis by monitoring the production of ribosomes, and that this monitoring is apparently carried out through their activity in the initiation (and perhaps subsequent steps) of translation.     

Anatomy of Escherichia coli ribosome binding sites

During translational initiation in prokaryotes, the 3' end of the 16S rRNA binds to a region just upstream of the initiation codon. The relationship between this Shine-Dalgarno (SD) region and the binding of ribosomes to translation start-points has been well studied, but a unified mathematical connection between the SD, the initiation codon and the spacing between them has been lacking. Using information theory, we constructed a model that treats these three components uniformly by assigning to the SD and the initiation region (IR) conservations in bits of information, and by assigning to the spacing an uncertainty, also in bits. To build the model, we first aligned the SD region by maximizing the information content there. The ease of this process confirmed the existence of the SD pattern within a set of 4122 reviewed and revised Escherichia coli gene starts. This large data set allowed us to show graphically, by sequence logos, that the spacing between the SD and the initiation region affects both the SD site conservation and its pattern. We used the aligned SD, the spacing, and the initiation region to model ribosome binding and to identify gene starts that do not conform to the ribosome binding site model. A total of 569 experimentally proven starts are more conserved (have higher information content) than the full set of revised starts, which probably reflects an experimental bias against the detection of gene products that have inefficient ribosome binding sites. Models were refined cyclically by removing non-conforming weak sites. After this procedure, models derived from either the original or the revised gene start annotation were similar. Therefore, this information theory-based technique provides a method for easily constructing biologically sensible ribosome binding site models. Such models should be useful for refining gene-start predictions of any sequenced bacterial genome.     

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.     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.     

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.