Requirement of upstream Hfq-binding (ARN)x elements in glmS and the Hfq C-terminal region for GlmS upregulation by sRNAs GlmZ and GlmY{dagger}

Hfq is an important RNA-binding protein that helps bacteria adapt to stress. Its primary function is to promote pairing between trans-acting small non-coding RNAs (sRNAs) and their target mRNAs. Identification of essential Hfq-binding motifs in up-stream regions of rpoS and fhlA led us to ask the question whether these elements are a common occurrence among other Hfq-dependent mRNAs as well. Here, we confirm the presence of a similar (ARN)(x) motif in glmS RNA, a gene controlled by two sRNAs (GlmZ and GlmY) in an Hfq-dependent manner. GlmZ represents a canonical sRNA:mRNA pairing system, whereas GlmY is non-canonical, interfacing with the RNA processing protein YhbJ. We show that glmS interacts with both Hfq-binding surfaces in the absence of sRNAs. Even though two (ARN)(x) motifs are present, using a glmS:gfp fusion system, we determined that only one specific (ARN)(x) element is essential for regulation. Furthermore, we show that residues 66-72 in the C-terminal extension of Escherichia coli Hfq are essential for activation of GlmS expression by GlmY, but not with GlmZ. This result shows that the C-terminal extension of Hfq may be required for some forms of non-canonical sRNA regulation involving ancillary components such as additional RNAs or proteins.     

Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase

B?ck, August (Purdue University, Lafayette, Ind.), Lia Eidlic Faiman, and Frederick C. Neidhardt. Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase. J. Bacteriol. 92:1076-1082. 1966.-To test our conclusion that Escherichia coli mutant I-9 possesses a valyl soluble ribonucleic acid (sRNA) synthetase that functions in vivo at 30 C but not at 37 C, measurements were made by use of the periodate method, of the level of charged valyl sRNA in this strain. A shift of temperature from 30 to 40 C resulted in a rapid discharging of valyl sRNA coordinate with the cessation of protein synthesis; at the same time, other species of sRNA, such as those for leucine, became fully charged. Identical results were obtained with a derivative of I-9 with relaxed ribonucleic acid (RNA) control. When P1 phage were grown on wild cells and then used at low multiplicities of infection to transduce temperature-resistant growth into I-9, complete cotransduction of normal valyl sRNA synthetase occurred. By means of the interrupted-mating technique, the structural gene for valyl sRNA synthetase was located on the E. coli chromosome map and found to be near thr, one-fifth of the length of the chromosome removed from the structural genes for the isoleucine-valine biosynthetic enzymes. Therefore, (i) the major valyl sRNA synthetase activity of I-9 appears to be temperature-sensitive in vivo, (ii) relaxed amino acid control over RNA synthesis does not appear to be a consequence of a normal charging of sRNA with a substitute molecule, and (iii) one structural gene for valyl sRNA synthetase is located on the E. coli chromosome not closely linked to the cistrons for the valine-biosynthetic enzymes.     

Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis

It was recently discovered that the aarF gene in Providencia stuartii is required for coenzyme Q (CoQ) biosynthesis. Here we report that yigR, the Escherichia coli homologue of aarF, is ubiB, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the P. stuartii aarF and E. coli ubiB (yigR) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the E. coli strain AN59, which contains the ubiB409 mutant allele. Analysis of the mutation in the E. coli strain AN59 reveals no mutations within the ubiB gene, but instead shows the presence of an IS1 element at position +516 of the ubiE gene. The ubiE gene encodes a C-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5' gene in an operon containing ubiE, yigP, and ubiB. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1 mutation on the downstream ubiB gene. AN59 is complemented by a DNA segment containing the contiguous ubiE, yigP, and ubiB genes. Although transformation of AN59 with a DNA segment containing the ubiB coding region fails to restore CoQ biosynthesis, transformation with the ubiE coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the fre gene, previously considered to correspond to ubiB, was found not to be involved in CoQ biosynthesis. The ubiB gene is a member of a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.     

细菌非编码小RNA研究进展

细菌非编码小RNA(small non-coding RNA, sRNA)是一类长度在50~500个核苷酸, 不编码蛋白质的RNA。迄今, 在各种细菌中共发现超过150多种sRNA。它们通过碱基配对识别靶标mRNA, 在转录后水平调节基因的表达, 是细菌代谢、毒力和适应环境压力的重要调节因子。细菌sRNA的研究技术主要有基于生物信息学的计算机预测法和基于实验室的检测分析方法。这些方法所得到的sRNA都需要进行实验室确认, 然后再进一步通过各种实验手段研究其功能。     

大肠杆菌O157:H7的sRNA伴侣蛋白Ufq的基因克隆、表达及纯化

目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157:H7的sRNA伴侣蛋白Hfq.方法:利用PCR方法从EHEC O157:H7基因组中扩增出基因hfq,并插入含6xHis标签序列的原核表达载体pET28a(+)的多克隆位点中,构建重组表达质粒pET28a(+)-hfq,以重组质粒转化大肠杆菌BL21(DE3)...     

Small RNA identification in Enterobacteriaceae using synteny and genomic backbone retention

Genomic screens for small RNA candidates in Enterobacteriacae genomes were carried out with existing small RNA sequences, conserved flanking genes, and genomic backbone information. The small RNA sequences and contexts from E. coli K12 formed the basis of the search. Sequence identity identified 117 additional small RNA homologs in related genomes. Motifs of continuous sequence stretches added another 48 sRNA regions, termed partial homologs. However, this study is unique in identifying 160 nonhomologous sRNA loci in related genomes based on the conserved flanking gene synteny and the backbone retention information obtained from KEGG-SSDB. Gene synteny and genomic backbone continuity were observed to be correlated with all of the sRNAs in related genomes. This search is the first of its kind toward identification of functionally important regions using gene order and back-bone information. A disruption in flanking gene order or genomic backbone indicates a possible hotspot for alien gene pool integration. This study reports both occurrence of multiple copies of a sRNA and co-occurrence of different sRNAs between a pair of conserved flanking genes. In general, synteny and genomic backbone retention information can be added as additional search criteria toward the design of precise bioinformatics tools for sRNA, gene identification, and gene functional annotations in related genomes.     

Conservation analysis of small RNA genes in Escherichia coli

MOTIVATION: Small RNA (sRNA) genes in Escherichia coli have been in focus recently, as 44 out of 55 experimentally confirmed sRNA genes have been precisely located in the genome. The object of this study is to analyze quantitatively the conservation of these sRNA genes and compare it with the conservation of protein-encoding genes, function-unknown regions and tRNA genes. RESULTS: The results show that within an evolutionary distance of 0.26, both sRNA genes and protein-encoding genes display a similar tendency in their degrees of conservation at the nucleotide level. In addition, the conservation of sRNA genes is much stronger than function-unknown regions, but much weaker than tRNA genes. Based on the conservation of studied sRNA genes, we also give clues to estimate the total number of sRNA genes in E.coli. SUPPLEMENTARY INFORMATION: Supplementary information is available at http://www.bioinfo.org.cn/SM/sRNAconservation.htm     

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.     

大肠杆菌yigP基因转录调控序列的鉴定

【目的】调查yigP基因启动子的活性,并对该转录调控序列进行分析。【方法】以lacZ为报告基因,克隆启动子片段至启动子探针质粒中,通过检测β-半乳糖苷酶活性判断启动子活性,并通过克隆一系列逐步缩短的启动子片段来确定启动子所在区域。利用定点突变技术,对启动子的重要序列进行定点突变,调查其对启动子活性的影响。【结果】确定了yigP基因启动子的区域,鉴定了启动子的-10区和-35区,并发现了启动子上游存在一个负调控序列,对该序列进行了初步的研究显示其中部分序列是这种负调控作用的核心序列。【结论】对yigP基因的转录调控序列进行了鉴定,丰富了我们对基因转录调控的认识。     

Effect of amino acid starvation on glucose transport in two archaeal organisms

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.     

Synthesis of Soluble Ribonucleates in Escherichia coli Infected with Bacteriophage R17

Measurements were made on the level of synthesis of soluble ribonucleates (sRNA) in Escherichia coli K-12 infected with the ribonucleic acid (RNA)-containing bacteriophage R17. Little or no decrease in sRNA synthesis was found to occur under conditions of infection which result in a 70 to 80% inhibition of ribosomal RNA synthesis.