Pascale Romby

Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell‐wall‐associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII‐mediated inhibition of translation and degradation of the stable spa mRNA by the double‐strand‐specific endoribonuclease III (RNase III). The 3′ end domain of RNAIII, partially complementary to the 5′ part of spa mRNA, efficiently anneals to spa mRNA through an initial loop–loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.

Antisense RNAs in bacteria and their genetic elements.

Antisense RNA-mediated regulation is widespread in bacteria. Most antisense RNA control systems have been found in plasmids, phages, and transposons. Fewer examples were identified in bacterial chromosomes. This chapter summarizes our current knowledge about antisense RNAs with respect to their occurrence, their biological roles, and their diverse mechanisms of action. Examples of cis- or trans-encoded antisense RNAs are discussed, and their properties compared. Most antisense RNAs are posttranscriptionally acting inhibitors of target genes, but a few examples of activator antisense RNAs are known. The implications of RNA structure on topologically and kinetically favored binding pathways are addressed, and solutions that have evolved to permit productive interactions between intricately folded RNAs are discussed. Finally, we describe how particular properties of individual antisense/target RNA systems match their respective biological roles.

The role of exportin‐t in selective nuclear export of mature tRNAs

Exportin‐t (Xpo‐t) is a vertebrate nuclear export receptor for tRNAs that binds tRNA cooperatively with GTP‐loaded Ran. Xpo‐t antibodies are shown to efficiently block tRNA export from Xenopus oocyte nuclei suggesting that it is responsible for at least the majority of tRNA export in these cells. We examine the mechanism by which Xpo‐t–RanGTP specifically exports mature tRNAs rather than other forms of nuclear RNA, including tRNA precursors. Chemical and enzymatic footprinting together with phosphate modification interference reveals an extensive interaction between the backbone of the TΨC and acceptor arms of tRNAPhe and Xpo‐t–RanGTP. Analysis of mutant or precursor tRNA forms demonstrates that, aside from these recognition elements, accurate 5′ and 3′ end‐processing of tRNA affects Xpo‐t–RanGTP interaction and nuclear export, while aminoacylation is not essential. Intron‐containing, end‐processed, pre‐tRNAs can be bound by Xpo‐t–RanGTP and are rapidly exported from the nucleus if Xpo‐t is present in excess. These results suggest that at least two mechanisms are involved in discrimination of pre‐tRNAs and mature tRNAs prior to nuclear export.

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation.

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism.

Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem.

Threonyl-tRNA synthetase, a class II synthetase, uses a unique zinc ion to discriminate against the isosteric valine at the activation step. The crystal structure of the enzyme with an analog of seryl adenylate shows that the noncognate serine cannot be fully discriminated at that step. We show that hydrolysis of the incorrectly formed ser-tRNA(Thr) is performed at a specific site in the N-terminal domain of the enzyme. The present study suggests that both classes of synthetases use effectively the ability of the CCA end of tRNA to switch between a hairpin and a helical conformation for aminoacylation and editing. As a consequence, the editing mechanism of both classes of synthetases can be described as mirror images, as already seen for tRNA binding and amino acid activation.

Small RNAs in Bacteria and Archaea: Who They Are, What They Do, and How They Do It

Small RNAs are ubiquitously present regulators in all kingdoms of life. Most bacterial and archaeal small RNAs (sRNAs) act by antisense mechanisms on multiple target mRNAs, thereby globally affecting essentially any conceivable trait-stress responses, adaptive metabolic changes, virulence etc. The sRNAs display many distinct mechanisms of action, most of them through effects on target mRNA translation and/or stability, and helper proteins like Hfq often play key roles. Recent data highlight the interplay between posttranscriptional control by sRNAs and transcription factor-mediated transcriptional control, and cross talk through mutual regulation of regulators. Based on the properties that distinguish sRNA-type from transcription factors-type control, we begin to glimpse why sRNAs have evolved as a second, essential layer of gene regulation. This review will discuss the prevalence of sRNAs, who they are, what biological roles they play, and how they carry out their functions.

A structural view of translation initiation in bacteria

Abstract.The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNAfMet to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation.

Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

The Staphylococcus aureus RNome and its commitment to virulence.

Staphylococcus aureus is a major human pathogen causing a wide spectrum of nosocomial and community-associated infections with high morbidity and mortality. S. aureus generates a large number of virulence factors whose timing and expression levels are precisely tuned by regulatory proteins and RNAs. The aptitude of bacteria to use RNAs to rapidly modify gene expression, including virulence factors in response to stress or environmental changes, and to survive in a host is an evolving concept. Here, we focus on the recently inventoried S. aureus regulatory RNAs, with emphasis on those with identified functions, two of which are directly involved in pathogenicity.