scholarly journals Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1000-1008 ◽  
Author(s):  
Andreas Licht ◽  
Peggy Freede ◽  
Sabine Brantl
Keyword(s):  
Rna Polymerase ◽  
Antisense Rna ◽  
Transcriptional Repressor ◽  
Broad Host Range ◽  
Dnase I Footprinting ◽  
C Terminus ◽  
Polymerase Binding ◽  
Rnap Binding ◽  
Competition Assays

CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10- to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR–operator complexes are 18-fold less stable than RNA polymerase (RNAP)–pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO4 footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.

scholarly journals The C Terminus of ς32 Is Not Essential for Degradation by FtsH

2001 ◽  
Vol 183 (20) ◽  
pp. 5911-5917 ◽  
Author(s):  
Toshifumi Tomoyasu ◽  
Florence Arsène ◽  
Teru Ogura ◽  
Bernd Bukau
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Recognition Sequence ◽  
Mutant Proteins ◽  
C Terminus ◽  
Polymerase Binding ◽  
Stress Dependent

ABSTRACT A key step in the regulation of heat shock genes inEscherichia coli is the stress-dependent degradation of the heat shock promoter-specific ς32 subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including ς32, as degradation signals for AAA proteases. We investigated the role of the C terminus of ς32 in FtsH-dependent degradation by analysis of C-terminally truncated ς32 mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of ς32 did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of ς32 was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated ς32 mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of ς32 in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.

scholarly journals Nrg1 Is a Transcriptional Repressor for Glucose Repression of STA1 Gene Expression inSaccharomyces cerevisiae

1999 ◽  
Vol 19 (3) ◽  
pp. 2044-2050 ◽  
Author(s):  
Seok Hee Park ◽  
Sang Seok Koh ◽  
Jae Hwan Chun ◽  
Hye Jin Hwang ◽  
Hyen Sam Kang
Keyword(s):  
Gene Expression ◽  
Zinc Finger Protein ◽  
Glucose Repression ◽  
Transcriptional Repressor ◽  
Dependent Manner ◽  
Expression Of Genes ◽  
Dnase I Footprinting ◽  
Genes Encoding

ABSTRACT Expression of genes encoding starch-degrading enzymes is regulated by glucose repression in the yeast Saccharomyces cerevisiae. We have identified a transcriptional repressor, Nrg1, in a genetic screen designed to reveal negative factors involved in the expression of STA1, which encodes a glucoamylase. TheNRG1 gene encodes a 25-kDa C2H2zinc finger protein which specifically binds to two regions in the upstream activation sequence of the STA1 gene, as judged by gel retardation and DNase I footprinting analyses. Disruption of theNRG1 gene causes a fivefold increase in the level of theSTA1 transcript in the presence of glucose. The expression of NRG1 itself is inhibited in the absence of glucose. DNA-bound LexA-Nrg1 represses transcription of a target gene 10.7-fold in a glucose-dependent manner, and this repression is abolished in bothssn6 and tup1 mutants. Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro. These findings indicate that Nrg1 acts as a DNA-binding repressor and mediates glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex.

scholarly journals iRAPs curb antisense transcription in E. coli

2019 ◽  
Vol 47 (20) ◽  
pp. 10894-10905 ◽  
Author(s):  
Andrés Magán ◽  
Fabian Amman ◽  
Fatinah El-Isa ◽  
Natascha Hartl ◽  
Ilya Shamovsky ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Antisense Transcription ◽  
Rna Aptamers ◽  
Antisense Strand ◽  
Genomic Context ◽  
Reporter System ◽  
E Coli ◽  
Polymerase Binding ◽  
In Cis

Abstract RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.

scholarly journals FleQ, the Major Flagellar Gene Regulator in Pseudomonas aeruginosa, Binds to Enhancer Sites Located Either Upstream or Atypically Downstream of the RpoN Binding Site

2002 ◽  
Vol 184 (19) ◽  
pp. 5251-5260 ◽  
Author(s):  
Jeevan Jyot ◽  
Nandini Dasgupta ◽  
Reuben Ramphal
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Binding Sites ◽  
Direct Interaction ◽  
Significant Loss ◽  
Transcription Start Sites ◽  
Dnase I Footprinting ◽  
Gel Shift Assay ◽  
Polymerase Binding

ABSTRACT In Pseudomonas aeruginosa, flagellar genes are regulated in a cascade headed by FleQ, an NtrC/NifA-type activator. FleQ and RpoN positively regulate expression of flhA, fliE, fliL, and fleSR genes, among others. Direct interaction of FleQ with flhA, fliE, fliL, and fleSR promoters was demonstrated by gel shift assay, along with experiments to conclusively determine the specificity of its binding. DNase I footprinting was performed to determine the FleQ binding sites on flhA, fliE, fliL, and fleSR promoters. No sequence conservation among these binding sites was observed. Primer extension analysis revealed the transcription start sites (TSSs) to be localized above the FleQ binding sites in flhA, fliE, and fliL promoters. Analysis of the above data revealed FleQ binding to be in the leader sequence of these promoters, whereas FleQ binding was 67 bp upstream of the TSS in the fleSR promoter. Mutagenesis of the FleQ binding site in the flhA promoter confirmed its functionality in vivo. Deletion of the flhA promoter upstream of the RNA polymerase binding site did not result in a significant loss of promoter activity. These results point to two modes of regulation by an NtrC-type regulator in the flagellar hierarchy in P. aeruginosa, the first being the typical model of activation from a distance via looping in the fleSR promoter and the second involving flhA, fliE, and fliL promoters, where FleQ binds in the downstream vicinity of the promoter and activates transcription without looping.

scholarly journals An archaebacterial promoter sequence assigned by RNA polymerase binding experiments

1989 ◽  
Vol 35 (1) ◽  
pp. 30-35 ◽  
Author(s):  
Michael Thomm ◽  
Günter Wich ◽  
James W. Brown ◽  
Gerhard Frey ◽  
Bruce A. Sherf ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Specific Binding ◽  
Promoter Sequence ◽  
Rrna Genes ◽  
Protein Encoding ◽  
Nuclease Digestion ◽  
Polymerase Binding

To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA polymerase of Methanococcus vannielii were performed. The enzyme binds specifically both at protein-encoding (hisA and methyl CoM reductase, component C) and tRNA–rRNA genes. The binding region of the RNA polymerase extends from 30 base pairs (bp) upstream (−30) to 20 bp downstream (+20) from the in vivo transcription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting traascription factors. The DNA segment protected from nuclease digestion by bound RNA polymerase contains an octanucleotide sequence centered at −25, which is conserved between the protein-encoding and the stable RNA genes. According to the specific binding of the enzyme to only DNA-fragments harbouring this motif, we propose the sequence TTTATATA as the major recognition signal of the Methanococcus RNA polymerase. Comparison of this motif with published archaebacterial DNA sequences revealed the presence of homologous sequences at the same location upstream of 36 genes. We therefore consider the overall consensus [Formula: see text] as a general element of promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of the Methanococcus vannielii RNA polymerase are unable to initiate transcription at the correct sites in vitro. Here we present first evidence for the possible existence of a transcription factor conferring the ability to the enzyme to initiate and terminate transcription specifically in vitro.Key words: promoter, footprint, TATA box, RNA polymerase, transcription.

scholarly journals Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase

2003 ◽  
Vol 77 (7) ◽  
pp. 4149-4159 ◽  
Author(s):  
Lu Gao ◽  
Hong Tu ◽  
Stephanie T. Shi ◽  
Ki-Jeong Lee ◽  
Miyuki Asanaka ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Subgenomic Replicon ◽  
C Terminus ◽  
Huh7 Cells

ABSTRACT To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

scholarly journals Repression of RNA Polymerase II Elongation In Vivo Is Critically Dependent on the C-Terminus of Spt5

PLoS ONE ◽  
2009 ◽  
Vol 4 (9) ◽  
pp. e6918 ◽  
Author(s):  
Hui Chen ◽  
Xavier Contreras ◽  
Yuki Yamaguchi ◽  
Hiroshi Handa ◽  
B. Matija Peterlin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
C Terminus

scholarly journals In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς54-Dependent Po Promoter

2001 ◽  
Vol 183 (9) ◽  
pp. 2842-2851 ◽  
Author(s):  
Chun Chau Sze ◽  
Andrew D. Laurie ◽  
Victoria Shingler
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Binding Sites ◽  
Integration Host Factor ◽  
Host Factor ◽  
Physical Interaction ◽  
Rich Region ◽  
Polymerase Binding

ABSTRACT Transcription from the Pseudomonas CF600-derived ς54-dependent promoter Po is controlled by the aromatic-responsive activator DmpR. Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro. In vivo, the Po promoter exhibits characteristics that typify many ς54-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output. IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites. In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription. This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at ς54 promoters such as aiding binding of the regulator or recruitment of ς54-RNA polymerase via UP element-like DNA. The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA. Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter.

scholarly journals Nucleoid openness profiling links bacterial genome structure to phenotype

2020 ◽  
Author(s):  
Mahmoud M Al-Bassam ◽  
Oriane Moyne ◽  
Nate Chapin ◽  
Karsten Zengler
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Structural Changes ◽  
Bacterial Genome ◽  
Genome Structure ◽  
Housekeeping Genes ◽  
Associated Proteins ◽  
Polymerase Binding

ABSTRACTGene expression requires specific structural alternations in the nucleoid structure to enable the access of the transcription machinery into the genomic DNA. In prokaryotes, DNA binding proteins, including nucleoid-associated proteins (NAPs) and transcription factors (TFs), drive the change in structure and gene expression. Currently, studies of global NAP and TF binding are often hindered by the lack of appropriate epigenomic tools. Here, we present POP-seq, a method that provides in vivo genome-wide openness profiles of the bacterial nucleoid. We demonstrate that POP-seq can be used to map the global in vivo protein-DNA binding events. Our results highlight a negative correlation between genome openness, compaction and transcription, suggesting that regions that are not accessible to Tn5 transposase are either too compacted or occupied by RNA polymerase. Importantly, we also show that the least open regions are enriched in housekeeping genes, while the most open regions are significantly enriched in genes important for fast adaptation to changing environment. Finally, we demonstrated that the genome openness profile is growth condition specific. Together, those results suggest a model where one can distinguish two types of epigenetic control: one stable, long-term silencing of highly compacted regions, and a second, highly responsive regulation through the dynamic competition between NAPs and RNA polymerase binding. Overall, POP-seq captures structural changes in the prokaryotic chromatin and provides condition-specific maps of global protein-DNA binding events, thus linking overall transcriptional and epigenetic regulation directly to phenotype.

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