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     

Archaeology and evolution of transfer RNA genes in the Escherichia coli genome

Transfer RNA genes tend to be presented in multiple copies in the genomes of most organisms, from bacteria to eukaryotes. The evolution and genomic structure of tRNA genes has been a somewhat neglected area of molecular evolution. Escherichia coli, the first phylogenetic species for which more than two different strains have been sequenced, provides an invaluable framework to study the evolution of tRNA genes. In this work, a detailed analysis of the tRNA structure of the genomes of Escherichia coli strains K12, CFT073, and O157:H7, Shigella flexneri 2a 301, and Salmonella typhimurium LT2 was carried out. A phylogenetic analysis of these organisms was completed, and an archaeological map depicting the main events in the evolution of tRNA genes was drawn. It is shown that duplications, deletions, and horizontal gene transfers are the main factors driving tRNA evolution in these genomes. On average, 0.64 tRNA insertions/duplications occur every million years (Myr) per genome per lineage, while deletions occur at the slower rate of 0.30 per million years per genome per lineage. This work provides a first genomic glance at the problem of tRNA evolution as a repetitive process, and the relationship of this mechanism to genome evolution and codon usage is discussed.     

A survey of small RNA-encoding genes in Escherichia coli

Small RNA (sRNA) molecules have gained much interest lately, as recent genome-wide studies have shown that they are widespread in a variety of organisms. The relatively small family of 10 known sRNA-encoding genes in Escherichia coli has been significantly expanded during the past two years with the discovery of 45 novel genes. Most of these genes are still uncharacterized and their cellular roles are unknown. In this survey we examined the sequence and genomic features of the 55 currently known sRNA-encoding genes in E.coli, attempting to identify their common characteristics. Such characterization is important for both expanding our understanding of this unique gene family and for improving the methods to predict and identify sRNA-encoding genes based on genomic information.     

A bioinformatics approach to identifying tail-anchored proteins in the human genome

Intracellular proteins with a carboxy-terminal transmembrane domain and the amino-terminus oriented toward the cytosol are known as 'tail-anchored' proteins. Tail-anchored proteins have been of considerable interest because several important classes of proteins, including the vesicle-targeting/fusion proteins known as SNAREs and the apoptosis-related proteins of the Bcl-2 family, among others, utilize this unique membrane-anchoring motif. Here, we use a bioinformatic technique to develop a comprehensive list of potentially tail-anchored proteins in the human genome. Our final list contains 411 entries derived from 325 unique genes. We also analyzed both known and predicted tail-anchored proteins with respect to the amino acid composition of the transmembrane segments. This analysis revealed a distinctive composition of the membrane anchor in SNARE proteins.     

Identifying Hfq-binding small RNA targets in Escherichia coli

The Hfq-binding small RNAs (sRNAs) have recently drawn much attention as regulators of translation in Escherichia coli. We attempt to identify the targets of this class of sRNAs in genome scale and gain further insight into the complexity of translational regulation induced by Hfq-binding sRNAs. Using a new alignment algorithm, most known negatively regulated targets of Hfq-binding sRNAs were identified. The results also show several interesting aspects of the regulatory function of Hfq-binding sRNAs.     

Integrating structure,bioinformatics, and enzymology to discover function: BioH,a new carboxylesterase from Escherichia coli

Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 A resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure.     

大肠杆菌Small RNA EsrE的转录调控

【背景】大肠杆菌中Small RNA EsrE调控琥珀酸脱氢酶的表达并影响细胞生长,对其调控机制的探究有利于加深EsrE对细胞生长影响的认识。【目的】探究大肠杆菌Small RNA EsrE的转录调控机制。【方法】通过双质粒报告系统筛选转录调控因子,并通过凝胶迁移实验(electrophoretic mobilityshiftassay,EMSA)和qRT-PCR研究方法验证转录调控因子。【结果】双质粒报告系统证明RNA聚合酶亚基σ~(32) (RpoH)上调P_(esrE),β-羟酰-ACP脱水酶(FabZ)下调PesrE。EMSA结果和体内实验显示RpoH结合P_(esrE)片段,FabZ不结合P_(esrE)片段。【结论】RpoH直接结合启动子序列参与调控,FabZ以其他方式间接参与Small RNA EsrE的转录调控。     

A quantitative approach to catabolite repression in Escherichia coli

A dynamic mathematical model was developed to describe the uptake of various carbohydrates (glucose, lactose, glycerol, sucrose, and galactose) in Escherichia coli. For validation a number of isogenic strains with defined mutations were used. By considering metabolic reactions as well as signal transduction processes influencing the relevant pathways, we were able to describe quantitatively the phenomenon of catabolite repression in E. coli. We verified model predictions by measuring time courses of several extra- and intracellular components such as glycolytic intermediates, EII-ACrr phosphorylation level, both LacZ and PtsG concentrations, and total cAMP concentrations under various growth conditions. The entire data base consists of 18 experiments performed with nine different strains. The model describes the expression of 17 key enzymes, 38 enzymatic reactions, and the dynamic behavior of more than 50 metabolites. The different phenomena affecting the phosphorylation level of EIIACrr, the key regulation molecule for inducer exclusion and catabolite repression in enteric bacteria, can now be explained quantitatively.     

Gross map location of Escherichia coli transfer RNA genes.

Chromosomal locations of Escherichia coli genes specifying more than 20 different transfer RNA species were determined by utilizing two different methods. One was based upon gene dosage effects caused by F′ factors. In 15 different F′ strains and their corresponding F^? strains, relative contents of individual tRNAs were measured after separating the tRNAs by two-dimensional polyacrylamide gel electrophoresis. Approximate doubling of the content of particular tRNA was found in individual F′ strains, as showing gross map location of the tRNA gene. The other method was based on the amplified synthesis of tRNAs occurring after prophage induction of λ lysogens. Synthesis of individual tRNAs was measured after the induction of λ phages integrated at five different bacterial sites. Characteristic overproduction of different tRNAs was observed in individual prophage strains. This finding also gave approximate map locations of tRNA genes close to the prophage sites. The mapping data obtained by the two methods were consistent with each other and also with the reported positions in the cases where previously mapped. On the basis of map location of the tRNA_f1^Met gene newly determined, the λ-transducing phage carrying the tRNA_f1^Met gene was found.     

A complementary bioinformatics approach to identify potential plant cell wall glycosyltransferase-encoding genes

Plant cell wall (CW) synthesizing enzymes can be divided into the glycan (i.e. cellulose and callose) synthases, which are multimembrane spanning proteins located at the plasma membrane, and the glycosyltransferases (GTs), which are Golgi localized single membrane spanning proteins, believed to participate in the synthesis of hemicellulose, pectin, mannans, and various glycoproteins. At the Carbohydrate-Active enZYmes (CAZy) database where e.g. glucoside hydrolases and GTs are classified into gene families primarily based on amino acid sequence similarities, 415 Arabidopsis GTs have been classified. Although much is known with regard to composition and fine structures of the plant CW, only a handful of CW biosynthetic GT genes-all classified in the CAZy system-have been characterized. In an effort to identify CW GTs that have not yet been classified in the CAZy database, a simple bioinformatics approach was adopted. First, the entire Arabidopsis proteome was run through the Transmembrane Hidden Markov Model 2.0 server and proteins containing one or, more rarely, two transmembrane domains within the N-terminal 150 amino acids were collected. Second, these sequences were submitted to the SUPERFAMILY prediction server, and sequences that were predicted to belong to the superfamilies NDP-sugartransferase, UDP-glycosyltransferase/glucogen-phosphorylase, carbohydrate-binding domain, Gal-binding domain, or Rossman fold were collected, yielding a total of 191 sequences. Fifty-two accessions already classified in CAZy were discarded. The resulting 139 sequences were then analyzed using the Three-Dimensional-Position-Specific Scoring Matrix and mGenTHREADER servers, and 27 sequences with similarity to either the GT-A or the GT-B fold were obtained. Proof of concept of the present approach has to some extent been provided by our recent demonstration that two members of this pool of 27 non-CAZy-classified putative GTs are xylosyltransferases involved in synthesis of pectin rhamnogalacturonan II (J. Egelund, B.L. Petersen, A. Faik, M.S. Motawia, C.E. Olsen, T. Ishii, H. Clausen, P. Ulvskov, and N. Geshi, unpublished data).     

Organization of the Escherichia coli genome

A review of literature data reveals that for the last years, the molecular biology techniques have been of an increasing use in the study of the Escherichia coli genome, having supplemented the standard genetic mapping. For the proper understanding of the Escherichia coli genome organization, recombinational events occurring in the course of evolution should be considered. The bacterial genome seems to carry traces of both "long-term" evolution, possibly responsible for appearance of the bacterial cell itself, and "current" evolution, consisting mainly of periodic genome entering by new plasmid-originated genes. It is supposed that in the process of stabilization within a genome, every new gene undergoes a stage of the "transgene", that is the gene situated in a transposon on the chromosome. In parallel with integration of new genes into the genome, some genes deleting should also take place. The formation of deletions could occur by unequal crossing over in segments of direct homologous repeats which seem to be ordinarily revealed in the experimental study of the tandem gene duplications.     

基于TCGA数据预测肿瘤代表性新抗原的一种生物信息学新方案

肿瘤的特异性基因突变是肿瘤免疫疗法的理想靶标,突变的基因在健康组织中缺乏表达,而且具有高度免疫原性,容易被免疫系统识别。肿瘤患者突变基因组的高度特异性使得个体化免疫治疗存在极大挑战,而每一种肿瘤都具有区别于其他肿瘤的代表性的基因突变特征,基于这些突变特征,有可能开发出特定肿瘤适用的免疫治疗策略。文中提出一个兼顾抗原胞内呈递和与胞外MHC分子结合能力的肿瘤新抗原预测策略,整体设计更为合理;相对于常规方法,能够大幅缩小实验验证的范围。基于该策略,利用TCGA数据库中多种肿瘤的基因突变数据进行肿瘤新抗原预测并预测到大量潜在的肿瘤新抗原。肿瘤新抗原的预测结果显示出肿瘤类型的特异性,并且在特定肿瘤数据集中能够覆盖20%-70%不等比例的肿瘤患者。文中提出的肿瘤新抗原预测方案在未来的肿瘤临床治疗上具有潜在的应用价值。     

Physical map of the seven ribosomal RNA genes of Escherichia coli.

Escherichia coli DNA was digested with restriction endonucleases BamHI, PstI, EcoRI, SalI, HindIII, XhoI, BglII, SmaI, HpaI and with selected double and triple combinations of the same enzymes. The digests were electrophoresed and hybridized with 32P-labelled ribosomal RNA by using the Southern blotting technique. The resulting bands could be arranged into seven groups, and it was possible to construct a unique physical map of the seven rRNA genes (operons) of the bacterial chromosome. Mapping information obtained on several transducing phages and recombinant plasmids carrying rRNA genes, and mapping data published in the literature helped to determine the final map. The results suggest that phage lambda daroE152 carries a "hybrid" rRNA gene which was probably formed by recombination between two different chromosomal rRNA genes.     

Sequence and characterization of the Escherichia coli genome between the ndk and gcpE genes

Abstract The region of the chromosome immediately upstream of the Escherichia coli gene gcpE has been cloned and sequenced. This region contains two functional open reading frames, orf 384 and orf 337, encoding proteins of 43082 and 36189 Da, respectively. Sequencing analysis (this paper) and the isolation of a DNA fragment containing a functional promoter (Talukder, A.A., Yanai, S., and Yamada, M. (1994) Biosci. Biotech. Biochem. 58, 117–120) indicate that orf 337 is in an operon with gcpE . The gene orf 384 is immediately downstream of the gene ndk , which encodes nucleoside diphosphate kinase.