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Query: UMLS:C0009443 (cold)
 92,137 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The suhB gene of Escherichia coli has been defined by its mutant allele that suppresses other mutants in secY, rpoH, dnaB, and era. The suhB mutant by itself is cold sensitive, and is shown to have defects in protein synthesis. Starting with the suhB cold-sensitive mutant, cold-resistant suppressors were isolated. These suppressors mapped to the gene rnc encoding RNase III (a double-strand RNA-processing enzyme), and restored normal protein synthesis to the suhB mutants. Two known rnc mutations, rnc70 or rnc105, both defective in RNA cleavage activity, similarly restored growth of suhB. These rnc mutations did not alter the level of suhB expression. These results suggest that wild-type RNase III exerts a lethal effect on E coli upon depletion of SuhB at low temperatures. One explanation is to assume that the double-strand RNA-processing activity of RNase III itself is potentially lethal to E coli and the normal function of SuhB modulates the lethal action of RNase III.
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PMID:Lethal double-stranded RNA processing activity of ribonuclease III in the absence of suhB protein of Escherichia coli. 858 60

When Escherichia coli cells are shifted to low temperatures (e.g. 15 degrees C), growth halts while the 'cold shock response' (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3'-exoribonuclease and component of the RNA degradosome. At 37 degrees C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold-temperature induction involves several post-transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold-temperature adaptation, whereas lacZ(+) translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15 degrees C. However, unlike the lacZ(+) mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA-specific decay process is initiated during adaptation.
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PMID:Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation. 1112 93

The effects of base change mutations in a highly conserved sequence (boxC) within the leader of bacterial ribosomal RNAs (rRNAs) was studied. The boxC sequence preceding the 16S rRNA structural gene constitutes part of the RNase III processing site, one of the first cleavage sites on the pathway to mature 16S rRNA. Moreover, rRNA leader sequences facilitate correct 16S rRNA folding, thereby assisting ribosomal subunit formation. Mutations in boxC cause cold sensitivity and result in 16S rRNA and 30S subunit deficiency. Strains in which all rRNA operons are replaced by mutant transcription units are viable. Thermodynamic studies by temperature gradient gel electrophoresis reveal that mutant transcripts have a different, less ordered structure. In addition, RNA secondary structure differences between mutant and wild-type transcripts were determined by chemical and enzymatic probing. Differences are found in the leader RNA sequence itself but also in structurally important regions of the mature 16S rRNA. A minor fraction of the rRNA transcripts from mutant operons is not processed by RNase III, resulting in a significantly extended precursor half-life compared to the wild-type. The boxC mutations also give rise to a new aberrant degradation product of 16S rRNA. This intermediate cannot be detected in strains lacking RNase III. Together the results indicate that the boxC sequence, although important for RNase III processing, is likely to serve additional functions by facilitating correct formation of the mature 16S rRNA structure. They also suggest that quality control steps are acting during ribosome biogenesis.
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PMID:Effects of base change mutations within an Escherichia coli ribosomal RNA leader region on rRNA maturation and ribosome formation. 1150 77

We have examined the expression of pnp encoding the 3'-5'-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5' ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.
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PMID:Organization and expression of the polynucleotide phosphorylase gene (pnp) of Streptomyces: Processing of pnp transcripts in Streptomyces antibioticus. 1512 78

RNA interference (RNAi) has been greatly exploited in recent years as an increasingly effective tool to study gene function by gene silencing. The introduction of exogenous double-stranded RNA (dsRNA) into a cell can trigger this gene silencing process. An RNase III family enzyme, Dicer, initiates silencing by releasing approximately 20 base duplexes, with 2- nucleotide 3' overhangs called siRNAs. The RNAi pathway also mediates the function of endogenous, noncoding regulatory RNAs called miRNAs. Both miRNAs and siRNAs guide substrate selection by similar if not identical effector complexes called RISCs. These contain single-stranded versions of the small RNA and additional protein components. Of those, the signature element, at the heart of all RISCs, is a member of the Argonaute family of proteins. Our structural and biochemical studies on Argonaute identified this protein as Slicer, the enzyme in RISC that cleaves the mRNA as directed by the siRNA. The role of the Argonautes as Slicers and non-Slicers is discussed.
Cold Spring Harb Symp Quant Biol 2006
PMID:The Argonautes. 1738 Dec 82

The broad cellular actions of RNase III family enzymes include ribosomal RNA (rRNA) processing, mRNA decay, and the generation of noncoding microRNAs in both prokaryotes and eukaryotes. Here we report that YmdB, an evolutionarily conserved 18.8-kDa protein of Escherichia coli of previously unknown function, is a regulator of RNase III cleavages. We show that YmdB functions by interacting with a site in the RNase III catalytic region, that expression of YmdB is transcriptionally activated by both cold-shock stress and the entry of cells into stationary phase, and that this activation requires the sigma-factor-encoding gene, rpoS. We discovered that down-regulation of RNase III activity occurs during both stresses and is dependent on YmdB production during cold shock; in contrast, stationary-phase regulation was unperturbed in YmdB-null mutant bacteria, indicating the existence of additional, YmdB-independent, factors that dynamically regulate RNase III actions during normal cell growth. Our results reveal the previously unsuspected role of ribonuclease-binding proteins in the regulation of RNase III activity.
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PMID:YmdB: a stress-responsive ribonuclease-binding regulator of E. coli RNase III activity. 1914 81

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.
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PMID:Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region. 2141 43

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III-mediated cleavage in the 5' untranslated region (5'UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5'UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.
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PMID:Global regulatory functions of the Staphylococcus aureus endoribonuclease III in gene expression. 2276 86

Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.
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PMID:Nus transcription elongation factors and RNase III modulate small ribosome subunit biogenesis in Escherichia coli. 2319 53

High-throughput sequencing (HTS) methods can provide short sequence reads for many millions of individual molecules in a sample, allowing the use of sequencing to measure the abundance of RNA molecules. To quantify the amount of a particular sequence in a sample of large RNAs (e.g., mRNAs), it is important to fragment the RNA into short pieces that can be ligated to oligonucleotides that allow polymerase chain reaction (PCR) amplification and sequencing. The most desired end structure of RNA for such ligation steps is a 5' phosphate and a 3' OH. Thus, enzymes that leave these groups after cleavage are of particular utility, avoiding the need to dephosphorylate the 3' end with phosphatases or phosphorylate the 5' end with kinase before proceeding. One such enzyme, RNase III, is widely available. Although it primarily cuts duplex RNA, this specificity is salt- and concentration-dependent, and many RNAs that lack strong extended duplexes are nonetheless susceptible to cleavage at many spots. RNA fragmentation by RNase III does not seem to grossly affect the distribution of RNA sequencing reads. Thus, it has become a standard method for creating nominally representative pools of transcriptome sequences with 5' phosphates and 3' OH for library construction. Three steps in preparing fragmented transcriptome RNA for sequencing library construction are described here: (1) fragmenting the RNA with RNase III to the extent that ~60-100-nucleotide fragments are created, (2) purifying the RNA from the RNase III reaction, and (3) analyzing the digestion products for their suitability in library production.
Cold Spring Harb Protoc 2013 May 01
PMID:Fragmentation of whole-transcriptome RNA using E. coli RNase III. 2363 72


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