Effect of promoter sequence, template superhelicity and E. coli DNA-binding protein (ssb) on transcription by bacteriophage N4 virion encapsidated RNA polymerase

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Statementby Peter George Markiewicz.
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LC ClassificationsMicrofilm 85/420 (Q)
The Physical Object
FormatMicroform
Paginationxii, 280 leaves
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Open LibraryOL2689332M
LC Control Number85890415

Abstract. Starting from a synthetic E. coli promoter with the consensus sequences at and regions, a sequence CAT frequently occurred in the RNA start points of natural promoters was introduced in the downstream of the consensus sequences, and the sequences around the RNA start points as well as the relative positions of CAT from the consensus sequences were by:   Other promoters of the same class, notably the E.

coli fis promoter and the S. typhimurium hisR promoter 〚13〛, 〚49〛 are similarly only active on highly supercoiled templates. Template superhelicity and E. coli DNA-binding protein book the absence of any compensating mechanisms the fis -dependent general reduction in negative superhelicity might be expected to reduce the activity of promoters Cited by: The single-stranded DNA-binding protein of Escherichia coli.

Meyer RR(1), Laine PS. Author information: (1)Department of Biological Sciences, University of Cincinnati, Ohio The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and by: Shuichi Kusano, Quinquan Ding, Nobuyuki Fujita, Akira Ishihama.

Effect of DNA superhelicity on E. coli RNA polymerase binding to p R-derived promoters. Gel retardation analysis was used to indirectly estimate the affinity of E. coli RNA polymerase for the p R. The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus.

We have performed a computer analysis to study the prevalence of DNA static curvature in the regulatory regions of Escherichia coli, detecting a large number of operons with curved DNA fragments in their 5′ upstream regions.A statistical analysis reveals that all the global transcription factors identified so far in E.

coli have a tendency to regulate operons with curved DNA sequences in. Promoter strength (the promoter ability to initiate transcription) was measured with the help of fluorescent labelling method in microarray experiments on the total transcripts of E.

coli. Promoter strength was determined in arbitrary units reflecting the fluorescence intensity (Kanehisa et. The E. coli promoter consensus sequence Analysis of many E.

coli promoters has revealed that there are 3 conserved elements in the E. coli promoter: 35 sequence Centred 35 base pairs upstream of the start-point of transcription, this sequence element has the consensus sequence TTGACA sequence.

Lac promoter incorporated into the genome of E. coli allows for inducible expression of the T7 RNA polymerase by the normal transcription machinery in the cell.

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T7 promoter on the plasmid introduced into E. coli is placed in front of the DNA encoding the shRNA. The T7 RNA polymerase recognizes and transcribes from this promoter.

In this article, I review recent progress in studying protein-induced ΔLk by several sequence-specific DNA-binding proteins, such as E. template superhelicity and E.

coli DNA-binding protein book cAMP receptor protein (CRP) and lactose repressor (LacI). It was demonstrated recently that protein-induced ΔLk is an intrinsic property for sequence-specific DNA-binding proteins and does not correlate.

One such protein is the E. coli catabo- lite gene activator protein (CAP), a protein containing a helix-turn-helix DNA-binding domain. The dimeric unit binds to approximately 30 base pairs, making sequence- specific contacts in the major groove at a sequencedo- cated 5 bp from the midpoint of the binding site and, concomitantly bending the.

The effect of superhelicity of DNA templates on transcription is well documented in several cases. which corresponds to E. coli promoters recognized as was the beginning of a coding. Two host factors are required for N4 early transcription: Escherichia coli DNA gyrase (Falco et al.

) and E. coli single-stranded DNA–binding protein (EcoSSB) (Markiewicz et al. In vitro, supercoiled promoter-containing templates do not support virion RNA polymerase activity unless EcoSSB is present (Markiewicz et al. Olins PO, Nomura M. Regulation of the S10 ribosomal protein operon in E.

coli: nucleotide sequence at the start of the operon.

Description Effect of promoter sequence, template superhelicity and E. coli DNA-binding protein (ssb) on transcription by bacteriophage N4 virion encapsidated RNA polymerase PDF

Cell. Oct; 26 (2 Pt 2)– Burton ZF, Gross CA, Watanabe KK, Burgess RR. The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E.

coli K Cell. ator or the E. coli lactose or galactose repressors, strikingly stim-ulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase.

Also, the nature of the interaction of the specific DNA-binding protein with its recognition sequence, rather than the mass of the nucleoprotein complex, seems to be paramount. Finally, the absolute effect of a DNA-binding protein on DNA supercoiling during transcription can depend on the type of RNA polymerase used.

promoter-containing double-stranded DNAs when the template is supercoiled and Escherichia coli single-stranded DNA-binding protein (Eco SSB) is present.

We report that vRNAP–promoter. Promoter-specific binding was shown to be relatively insensitive to variations in the ionic strength of the incubation solution but dependent on the helical structure of the DNA. On the other hand, nonpromoter interior-site binding was Independent of the superhelicity of the DNA but extremely sensitive to changes in the ionic strength.

Bacteriophage N4 virion RNA polymerase transcrip- other single-stranded DNA–binding proteins cannot tion of double-stranded promoter-containing DNAs substitute (Markiewicz et al., ). Based on these re-requires supercoiled template and E.

coli single-sults. Ab initio motif detection and evaluation procedure. In order to predict putative AtoC binding sites, we implemented an ab initio motif detection procedure (Figure 1).This procedure involved an iterative pair-wise motif sampling process between the promoter of the atoDAEB operon, which was used as the initial qualified sequence, and a pool of E.

coli promoters (initial promoter set). Abstract. The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase.

Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. Promoter selectivity of Escherichia coli RNA polymerase: Effect of base substitutions in the promoter region on promoter strength January Nucleic Acids Research 18(24) Abstract. The heat-shock response (HSR), a universal cellular response to heat, is crucial for cellular adaptation.

In Escherichia coli, the HSR is mediated by the alternative σ factor, σ To determine its role, we used genome-wide expression analysis and promoter validation to identify genes directly regulated by σ 32 and screened ORF overexpression libraries to identify σ 32 inducers.

Production of recombinant proteins in Escherichia coli Wolfgang Schumann1 and Luis Carlos S. Ferreira2 1University of Bayreuth, Institute of Genetics, Bayreuth, Germany. 2Universidade de São Paulo, Instituto de Ciências Biomédicas, Departamento de Microbiologia, São Paulo, SP, Brazil.

Abstract Attempts to obtain a recombinant protein using prokaryotic expression systems can go from a. The σ factor σ 70 of E.

coli RNA polymerase acts not only in initiation, but also at an early stage of elongation to induce a transcription pause, and simultaneously to allow the phage λ gene Q transcription antiterminator to act. We identify the signal in DNA that induces early pausing to be a version of the σ 70 −10 promoter consensus, and we show that σ 70 is both necessary for.

The fragment encompassing the intrinsic curvature of the hns promoter region was obtained by PCR amplification of chromosomal DNA using QC2 (5′‐GAA GAC TGA AAG GTC G‐3′) and HC9 (5′‐CGC ACG AAG AGT ACG G‐3′) corresponding respectively to positions – and – of the hns sequence (Pon et al., ).

However, it is not known whether the abundant histone-like proteins present in E. coli, such as protein HU, would affect T7 RNAP activity in a similar way. HU is a basic, thermostable protein composed of two homologous subunits of 9 kDa which exists predominantly in E.

coli as a heterodimer (αβ) during the stationary phase (11, 50, 52). d. Up promoter mutations: increase the level of transcription.

Tend to make the promoter sequence more like the consensus. Down promoter mutations in the ‑35 sequence: decrease the rate of formation of the closed complex, indicating this is the sequence needed for intial recognition by the polymerase holoenzyme.

significantly alter the DNA-binding affinity of the protein (1). Thus, unlike other regulatory proteins, the binding of a coin-ducer molecule to a LysR protein activates transcription of a target gene by altering a property of the protein distinct from its DNA-binding activity. The ilvYC operon of Escherichia coli.

using E. coli. Optimizations in expression vector design, gene dosage, promoter strength (transcription regulation), mRNA stability, translation initiation and termination, E.

Details Effect of promoter sequence, template superhelicity and E. coli DNA-binding protein (ssb) on transcription by bacteriophage N4 virion encapsidated RNA polymerase FB2

coli host strain design, and codon usage have been performed, which result in significant enhancement of protein .available with E. coli signal peptide coding sequences at the 50 of MCS like p5 series (c5 series is for cytoplasmic expression) of pMAL vector, pET vector etc.

Solubility of recombinant protein in E. coli can be determined or checked with freely available software by conjugating the sequence of the fusion tag with the desired protein sequence.One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes.

We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them.

This effect was observed only in the absence of the integrase.