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		E.coli news vol. 13		2006.3.6
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>>>rbbA, ribosome-bound ATPase<<<
Nucleic Acids Res. 2006 Feb 22;34(4):1158-65.
Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes.
Xu J, Kiel MC, Golshani A, Chosay JG, Aoki H, Ganoza MC.
Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.

We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P- and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.

PMID: 16495476

>>>omrAB, OmpR regulated small RNAs A and B<<<
Mol Microbiol. 2006 Jan;59(1):231-47.
Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs.
Guillier M, Gottesman S.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Small non-coding RNAs that play important regulatory roles exist in numerous organisms. In Escherichia coli, about 60 small RNAs have been found and those that have been studied are involved in the response and adaptation to different stresses. RygA and RygB, two of these small RNAs, were identified on the basis of their conservation between different species and their ability to bind Hfq. They are adjacent on the chromosome and have sequence similarity at their 5' and 3' ends but distinct central regions, suggesting that they could regulate the expression of both common and distinct genes. A screen using a multicopy E. coli library led to identification of the response regulator OmpR and its associated sensor kinase EnvZ as positive regulators of rygA and rygB transcription. Therefore, RygA and RygB were renamed OmrA and OmrB respectively (for OmpR-regulated sRNAs A and B). When expressed at high levels, OmrA and OmrB RNAs negatively regulate the expression of sev eral genes encoding multiple outer membrane proteins, including cirA, fecA, fepA and ompT. Taken together, these data suggest that OmrA and OmrB participate in the regulation of outer membrane composition in response to environmental conditions.

PMID: 16359331

***additional finding***

>>>YhbH, YfiA, putative 90S/100S ribosome assmebly factor<<<
Genes Cells. 2005 Dec;10(12):1103-12.
Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli.
Ueta M, Yoshida H, Wada C, Baba T, Mori H, Wada A.
Department of Physics, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan.

During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named 'ribosomal hibernation,' RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild-type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P- and A-sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles.

PMID: 16324148

>>>csdE, CsdA binding protein, activate cysteine sulfinate desulfinase activity.<<<
J Biol Chem. 2005 Jul 22;280(29):26760-9. Epub 2005 May 18.
Analysis of the heteromeric CsdA-CsdE cysteine desulfurase, assisting Fe-S cluster biogenesis in Escherichia coli.
Loiseau L, Ollagnier-de Choudens S, Lascoux D, Forest E, Fontecave M, Barras F.
Laboratoire de Chimie Bacterienne, UPR-CNRS 9043, Institut de Biologie Structurale et Microbiologie, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.

Biogenesis of iron-sulfur (Fe-S) cluster-containing proteins relies on assistance of complex machineries. To date three systems, NIF, ISC, and SUF, were reported to allow maturation of Fe-S proteins. Here we report that the csdA-csdE (formally ygdK) genes of Escherichia coli constitute a sulfur-generating system referred to as CSD which also contributes to Fe-S biogenesis in vivo. This conclusion was reached by applying a thorough combination of both in vivo and in vitro strategies and techniques. Yeast two-hybrid analysis allowed us to show that CsdA and CsdE interact. Enzymology analysis showed that CsdA cysteine desulfurase activity is increased 2-fold in the presence of CsdE. Mass spectrometry analysis and site-directed mutagenesis showed that residue Cys-61 from CsdE acted as an acceptor site for sulfur provided by cysteine desulfurase activity of CsdA. Genetic investigations revealed that the csdA-csdE genes could act as multicopy suppressors of iscS mutation . Moreover, both in vitro and in vivo investigations pointed to a specific connection between the CSD system and quinolinate synthetase NadA.

PMID: 15901727 [PubMed - indexed for MEDLINE]

>>>YrdC, putative ribosome maturation factor<<<
Biochim Biophys Acta. 2005 Feb 14;1727(2):87-96.
The YrdC protein--a putative ribosome maturation factor.
Kaczanowska M, Ryden-Aulin M.
Department of Genetics, Microbiology and Toxicology (GMT), University of Stockholm, S-106 91 Stockholm, Sweden.

Release factor one (RF1) terminates protein synthesis in response to stop codons UAG and UAA. A mutant allele of RF1 causes temperature sensitive growth at 42 degrees C. We have earlier described the isolation of a suppressor of the temperature sensitive phenotype. The suppressor mutation is a small deletion in the open reading frame yrdC, and we have shown that the DeltayrdC mutation leads to immature 30S subunits and, as a consequence, to fewer translating ribosomes. YrdC is a small conserved protein with a dsRNA-binding surface. Here, we have characterized the YrdC protein. We show that the deletion leads to no production of functional protein, and we have indications that the YrdC protein might be essential in a wild type background. The protein is needed for the maturation of 16S rRNA, even though it does not interact tightly with either of the ribosomal subunits, or the 70S particles. The less effective maturation of rRNA affects the ribosomal feedback contro l, leading to an increase in expression from P1rrnB. We suggest that the function of the YrdC protein is to keep an rRNA structure needed for proper processing of 16S rRNA, especially at lower temperatures. This activity may require other factor(s). We suggest the gene be renamed rimN, and the mutant allele rimN141.

PMID: 15716138

>>>yabN, Putative ttranscriptional activator of sgrS small RNA gene<<<
Mol Microbiol. 2004 Nov;54(4):1076-89.
Involvement of a novel transcriptional activator and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system.
Vanderpool CK, Gottesman S.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

RyaA is a small non-coding RNA in Escherichia coli that was identified by its ability to bind tightly to the RNA chaperone Hfq. This study reports the role of RyaA in mediating the cellular response to glucose-specific phosphoenolypyruvate phosphotransferase system (PTS)-dependent phosphosugar stress. Aiba and co-workers have shown that a block in the metabolism of glucose 6-phosphate causes transient growth inhibition and post-transcriptional regulation of ptsG, encoding the glucose-specific PTS transporter. We found that RyaA synthesis was induced by a non-metabolizable glucose phosphate analogue and was necessary for relief of the toxicity of glucose phosphate stress. Expression of RyaA was sufficient to cause a rapid loss of ptsG mRNA, probably reflecting degradation of the message mediated by RyaA:ptsG pairing. The ryaA gene was renamed sgrS, for sugar transport-related sRNA. Expression of sgrS is regulated by a novel transcriptional activator, SgrR (formerly YabN), which has a putative DNA-binding domain and a solute-binding domain similar to those found in certain transport proteins. Our results suggest that under conditions of glucose phosphate accumulation, SgrR activates SgrS synthesis, causing degradation of ptsG mRNA. Decreased ptsG mRNA results in decreased production of glucose transport machinery, thus limiting further accumulation of glucose phosphate.

PMID: 15522088

>>>ybhO, Putative cardiolipin (CL) synthase<<<
Biochim Biophys Acta. 2000 Jan 17;1483(2):263-74.
A second Escherichia coli protein with CL synthase activity.
Guo D, Tropp BE.
Queens College CUNY, Department of Chemistry and Biochemistry, 65-30 Kissena Boulevard, Flushing, NY 11367, USA.

The Escherichia coli open reading frame f413, which has the potential to code for a polypeptide homologous to cardiolipin (CL) synthase, has been cloned. Its polypeptide product has a molecular mass of 48 kDa, is membrane-bound, and catalyzes CL formation but does not hydrolyze CL. A comparison of the sequences predicted for the polypeptides encoded by f413 and cls indicates that the N-terminal residues specified by cls may be unnecessary for CL synthase activity. Construction of a truncated cls gene and characterization of its polypeptide product have confirmed this conclusion.

PMID: 10634942

>>>rlmB, Ribosomal RNA large subunit methyltransferase (EC 2.1.1.-), (23S rRNA Gm2251 methyltransferase)<<<
J Bacteriol. 2001 Dec;183(23):6957-60.
The rlmB gene is essential for formation of Gm2251 in 23S rRNA but not for ribosome maturation in Escherichia coli.
Lovgren JM, Wikstrom PM.
Department of Molecular Biology, Umea University, S-901 87 Umea, Sweden.

In Saccharomyces cerevisiae, the rRNA Gm2270 methyltransferase, Pet56p, has an essential role in the maturation of the mitochondrial large ribosomal subunit that is independent of its methyltransferase activity. Here we show that the proposed Escherichia coli ortholog, RlmB (formerly YjfH), indeed is essential for the formation of Gm in position 2251 of 23S rRNA. However, a DeltarlmB mutant did not show any ribosome assembly defects and was not outgrown by a wild-type strain even after 120 cell mass doublings. Thus, RlmB has no important role in ribosome assembly or function in E. coli.

PMID: 11698387

>>>rbbA, ribosome-bound ATPase<<<
Biochimie. 1999 Dec;81(12):1097-108.
Identification of a ribosomal ATPase in Escherichia coli cells.
Kiel MC, Aoki H, Ganoza MC.
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canad.

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.

PMID: 10607404