The 5S rRNA maturase, ribonuclease M5, is a Toprim domain family member

The maturation of 5S ribosomal RNA in low G+C Gram-positive bacteria is catalyzed by a highly conserved, ∼190 residue, enzyme, called ribonuclease M5 (RNase M5). Sequence alignment had predicted that the N-terminal half of RNase M5 would consist of a Toprim domain, a protein fold found in type IA and type II topoisomerases, DnaG-like primases, OLD family nucleases and RecR proteins [L. Aravind, D. D. Leipe and E. V. Koonin (1998) Nucleic Acids Res., 26, 4205–4213]. Here, we present structural modelling data and a mutational analysis of RNase M5 that confirms this hypothesis. The N-terminal half of RNase M5 can be fitted to the Toprim domain of the DnaG catalytic core. Mutation of amino acid residues highly conserved among RNase M5 enzymes and members of the Toprim domain family showed that alteration of residues critical for topoisomerase and primase activity also had a dramatic effect on the cleavage of 5S rRNA precursor by RNase M5 both in vivo and in vitro. This suggests that the mechanisms of double-stranded RNA cleavage by RNase M5 and double-stranded DNA cleavage by members of the topoisomerase family are related

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3′ end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3′ extensions of the majority of CCA-less tRNAs

The crystal structure of Trz1, the long form RNase Z from yeast.

tRNAs are synthesized as precursor RNAs that have to undergo processing steps to become functional. Yeast Trz1 is a key endoribonuclease involved in the 3΄ maturation of tRNAs in all domains of life. It is a member of the β-lactamase family of RNases, characterized by an HxHxDH sequence motif involved in coordination of catalytic Zn-ions. The RNase Z family consists of two subfamilies: the short (250-400 residues) and the long forms (about double in size). Short form RNase Z enzymes act as homodimers: one subunit embraces tRNA with a protruding arm, while the other provides the catalytic site. The long form is thought to contain two fused β-lactamase domains within a single polypeptide. Only structures of short form RNase Z enzymes are known. Here we present the 3.1 Å crystal structure of the long-form Trz1 from Saccharomyces cerevisiae. Trz1 is organized into two β-lactamase domains connected by a long linker. The N-terminal domain has lost its catalytic residues, but retains the long flexible arm that is important for tRNA binding, while it is the other way around in the C-terminal domain. Trz1 likely evolved from a duplication and fusion of the gene encoding the monomeric short form RNase Z

Three Essential Ribonucleases—RNase Y, J1, and III—Control the Abundance of a Majority of Bacillus subtilis mRNAs

Bacillus subtilis possesses three essential enzymes thought to be involved in mRNA decay to varying degrees, namely RNase Y, RNase J1, and RNase III. Using recently developed high-resolution tiling arrays, we examined the effect of depletion of each of these enzymes on RNA abundance over the whole genome. The data are consistent with a model in which the degradation of a significant number of transcripts is dependent on endonucleolytic cleavage by RNase Y, followed by degradation of the downstream fragment by the 5′–3′ exoribonuclease RNase J1. However, many full-size transcripts also accumulate under conditions of RNase J1 insufficiency, compatible with a model whereby RNase J1 degrades transcripts either directly from the 5′ end or very close to it. Although the abundance of a large number of transcripts was altered by depletion of RNase III, this appears to result primarily from indirect transcriptional effects. Lastly, RNase depletion led to the stabilization of many low-abundance potential regulatory RNAs, both in intergenic regions and in the antisense orientation to known transcripts

RNA Processing and Degradation in Bacillus subtilis

This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli. A comparison of the genomes from the two organisms reveals that B. subtilis has a very different selection of RNases available for RNA maturation. Of 17 characterized ribonuclease activities thus far identified in E. coli and B. subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases. Some enzymes essential for cell viability in E. coli, such as RNase E and oligoribonuclease, do not have homologs in B. subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other. The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B. subtilis mRNAs. The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay. While RNase E binds to the 5′ end and catalyzes the rate-limiting cleavage of the majority of E. coli RNAs by looping to internal sites, the equivalent nuclease in B. subtilis, although not yet identified, is predicted to scan or track from the 5′ end. RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B. subtilis mRNAs appears incapable of direct entry. Thus, unlike E. coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5′ end can have very long half-lives even if the RNA is not protected by translation

Contrôle de l'activité de l'exoribonucléase 5'-3' Xrn1 par Dcs1 et conséquences physiologiques chez S. Cerevisiae

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

SURVEY AND SUMMARY: The phylogenetic distribution of bacterial ribonucleases

Ribonucleases play key, often essential, roles in cellular metabolism. Nineteen ribonuclease activities, from 22 different proteins, have so far been described in bacteria, the majority of them from either Escherichia coli or Bacillus subtilis. Here we examine the phylogenetic distribution of all of these ribonucleases in 50 eubacterial and archaeal species whose genomes have been completely sequenced, with particular emphasis on the endoribonucleases. Although some enzymes are very highly conserved throughout evolution, there appears to be no truly universal ribonuclease. While some organisms, like E.coli, have a large selection of ribonucleases, many with overlapping functions, others seem to have relatively few or have many that remain to be discovered

Biogénèse du ribosome chez les procaryotes (étude de la protéine ribosomique L20 d' Escherichia coli et de la maturase M5 de Bacillus subtilis)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Maturation of 23S rRNA in Bacillus subtilis in the Absence of Mini-III▿

23S rRNA maturation in Bacillus subtilis is catalyzed by the recently characterized enzyme Mini-RNase-III. Mini-III is dispensable, however, and 23S rRNA is matured by other ribonucleases in strains lacking this enzyme. Here we show that these RNases are the 5′-to-3′ exoribonuclease RNase J1 and the 3′-to-5′ exoribonucleases, principally RNase PH and YhaM