Chisato Ushida

Requirement of transfer‐messenger RNA for the growth of Bacillus subtilis under stresses

Bacterial transfer‐messenger RNA (tmRNA, 10Sa RNA) is involved in a trans‐translation reaction which contributes to the degradation of incompletely synthesized peptides and to the recycling of stalled ribosomes. However, its physiological role in the cell remains elusive. In this study, an efficient system for controlling the expression of the gene for tmRNA (ssrA), as well as a tmRNA gene‐defective strain (ssrA::cat), were constructed in Bacillus subtilis. The effects of tmRNA on the growth of the cells were investigated under various physiological culture conditions using these strains.

Detection of tmRNA-mediated trans-translation products in Bacillus subtilis.

Bacterial tmRNA (10Sa RNA) is involved in a trans‐translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes. To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo trans‐translation.

Interaction of 10Sa RNA with ribosomes in Escherichia coli

10Sa RNA is a bacterial small stable RNA, in which the 5′‐ and 3′‐end sequences are folded into a tRNA‐like structure. The RNA accepts alanine in vitro, and interacts with 70S ribosomes in the cells. In this study, we examined the ribosome‐binding properties of Escherichia coli 10Sa RNA in vivo, and found that the aminoacylation ability of 10Sa RNA with alanine is necessary for the binding to 70S ribosomes. 10Sa RNA was also found to bind only to 70S monosomes and not to polysomes. Recently, E. coli 10Sa RNA was suggested to be used as mRNA for tag peptides, which were found to attach to the C‐termini of truncated peptides synthesized in vivo. The present results are consistent with the ‘trans‐translation’ model, which has been proposed for tag‐peptide synthesis.

tmRNA-dependent trans-translation is required for sporulation in Bacillus subtilis

Spore formation in Bacillus subtilis is significantly impaired by the deletion of the gene for tmRNA (ssrA), which facilitates the trans‐translation reaction that rescues stalled ribosomes and degrades incompletely synthesized peptides. Microscopic analysis revealed that the sporulation of most ΔssrA cells is blocked after forespore formation. Expression analysis of lacZ‐fused genes directed by several RNA polymerase σ factors showed that the synthesis of active σK, encoded by the sigK gene, is predominantly inhibited in ΔssrA cells. The defect in σK synthesis is attributable to a defect in the skin element excision, which generates the sigK gene, caused in turn by reduced expression of SpoIVCA (recombinase) in ΔssrA cells.

Transcription and Translation

Most of our current knowledge of transcription and translation mechanisms in prokaryotes comes from the studies of Escherichia coli. These studies have provided detailed insights into many aspects of RNA and protein synthesis, including the components involved in these processes, individual reactions, regulation, kinetics etc. It is generally accepted that the mechanisms elucidated in the E. coli system can be extrapolated to those in other eubacterial species. The genetic and biochemical studies of mollicutes including the recent completion of the total genome sequences of Mycoplasma pneumoniae 21, M. genitalium 13, M. pulmonis 9a and Ureaplasma urealyticum 16, together with 214Kbp (about one/fifth of the total genome) sequence of M. capricolum 7, have revealed that the basic machineries and fundamental processes in transcription and translation are essentially the same as those of E. coli and other eubacteria. Since mycoplasmas belong to the gram-positive bacterial group having DNAs with a low G+C content, they share a common ancestor with bacilli, Clostridia, enterococci, lactobacilli, staphylococci and streptococci73. Therefore, many aspects of transcription and translation mechanisms of mycoplasmas are similar to those of the gram-positive rather than the gram-negative bacteria. However, several mycoplasma specific features, some of which are very important, have also been found.

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.

(p)ppGpp-dependent and -independent pathways for salt tolerance in Escherichia coli

Addition of some kinds of translation inhibitors targeting the ribosome such as kasugamycin to the culture medium as well as removal of a ribosome maturation factor or a ribosomal protein provides Escherichia coli cells with tolerance to high salt stress. Here, we found that another kind of translation inhibitor, serine hydroxamate (SHX), which induces amino acid starvation leading to (p)ppGpp production, also has a similar effect, but via a different pathway. Unlike kasugamycin, SHX was not effective in (p)ppGpp-null mutant cells. SHX and depletion of RsgA, a ribosome maturation factor, had an additive effect on salt tolerance, while kasugamycin or depletion of RsgA did not. These results indicate the presence of two distinct pathways, (p)ppGpp-dependent and -independent pathways, for salt tolerance of E. coli cell. Both pathways operate even in the absence of σ(S), an alternative sigma factor involved in the stationary phase or stress response. Hastened activation of the exocytoplasmic stress-specific sigma factor, σ(E), after salt shock was observed in the cells treated with SHX, as has been observed in the cells treated with a translation inhibitor or depleted of a ribosome maturation factor.

RNase P RNA ofMycoplasma capricolum

There are at least six small stable RNAs inMycoplasma capricolum cells besides tRNAs and rRNAs. One of them, MCS5 RNA, is a homolog of RNase P RNA. The predicted secondary structure of this RNA is essentially the same as that of other eubacterial RNase P RNAs. MCS5 RNA is more similar to the RNase P RNA ofB. subtilis than to that ofE. coli. This is consistent with previous conclusions that mycoplasmas are phylogenetically related to the low G+C Gram-positive bacterial group. The major substrates for MCS5 RNA must be the precursors of tRNAs. The precursor of MCS6 RNA, which is a homolog of theE. coli 10Sa RNA, may also be a substrate for the MCS5 RNA because this RNA has a tRNA-like structure at its 5′ and 3′ ends.

A small nucleolar RNA functions in rRNA processing in Caenorhabditis elegans.

CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.

Distribution of the MCS4 RNA genes in mycoplasmas belonging to the Mycoplasma mycoides cluster.

MCS4 RNA is one of the small stable RNAs found in Mycoplasma capricolum subsp. capricolum type strain California kid. This RNA has a sequence similarity to that of eukaryotic U6 snRNA. There are two genes encoding MCS4 RNA, designated mcs4a and mcs4b, in the genome. Homologous sequences of these genes were not found in databases of other bacterial sequences. We searched for MCS4 RNA and its genes in other bacteria by PCR and hybridization techniques. The results strongly suggested that this RNA exists only in a limited species of mycoplasmas belonging to the Mycoplasma mycoides cluster.