The α subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli

1994 ◽  
Vol 115 (1) ◽  
pp. 1-6
Author(s):  
V Bowrin
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Α Subunit

scholarly journals Cra-Dependent Transcriptional Activation of theicd Gene of Escherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 893-898 ◽  
Author(s):  
Jean-François Prost ◽  
Didier Nègre ◽  
Christelle Oudot ◽  
Katsuhiko Murakami ◽  
Akira Ishihama ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Α Subunit ◽  
P2 Promoter ◽  
Catabolite Repressor ◽  
A Site ◽  
Extension Analysis

ABSTRACT The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position −76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the α subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

scholarly journals The RNA Polymerase α Subunit from Sinorhizobium meliloti Can Assemble with RNA Polymerase Subunits from Escherichia coli and Function in Basal and Activated Transcription both In Vivo and In Vitro

2002 ◽  
Vol 184 (14) ◽  
pp. 3808-3814 ◽  
Author(s):  
Melicent C. Peck ◽  
Tamas Gaal ◽  
Robert F. Fisher ◽  
Richard L. Gourse ◽  
Sharon R. Long
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Sinorhizobium Meliloti ◽  
Core Promoter ◽  
Temperature Sensitive ◽  
Protein Sequence Analysis ◽  
Α Subunit ◽  
E Coli

ABSTRACT Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the α subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (α)-rplQ (L17) found in the α-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti α supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti α and E. coli β, β′, and σ subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti α functions are conserved in heterologous host E. coli even though the two α subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.

Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-subunit of RNA polymerase

2021 ◽  
Author(s):  
Helen Camakaris ◽  
Ji Yang ◽  
Tadashi Fujii ◽  
James Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Protein ◽  
Aromatic Amino Acids ◽  
Regulation Of Transcription ◽  
Plant Interactions ◽  
Α Subunit ◽  
Partial Suppression ◽  
K 12

A novel selection was developed for RpoA α-CTD mutants altered in activation by the TyrR regulatory protein of E. coli K-12. This allowed the identification of an aspartate to asparagine substitution in residue 250 (DN250) as an Act - mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and substitutions DR250, RE310 and RD310 were all shown to be defective in activation. None of these mutations caused defects in UP regulation. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. TyrR Mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77 and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act - tyrR mutants were isolated in an rpoA + strain, adding positions R77, D97, K101, D118, R119, R121 and E141 to known residues, S95 and D103, and defining the ‘activation patch’ on the NTD of TyrR. These results support a model for activation of TyrR-regulated genes where the ‘activation patch’ on the TyrR NTD interacts with the ‘TyrR-specific patch’ on the αCTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed, and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants, as Activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene TyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence, and important plant interactions.

scholarly journals Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

2007 ◽  
Vol 189 (23) ◽  
pp. 8430-8436 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Stable Complex ◽  
Tryptophan Residue ◽  
Specific Sequence ◽  
Bacterial Rna

ABSTRACT The heat shock sigma factor (σ32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, σ70. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ32, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium

2010 ◽  
Vol 88 (2) ◽  
pp. 529-539 ◽  
Author(s):  
Simon Stammen ◽  
Franziska Schuller ◽  
Sylvia Dietrich ◽  
Martin Gamer ◽  
Rebekka Biedendieck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacillus Megaterium ◽  
Protein Production ◽  
Rna Synthesis ◽  
Escherichia Coli Phage

scholarly journals Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
Author(s):  
Christine M. Beatty ◽  
Douglas F. Browning ◽  
Stephen J. W. Busby ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class I ◽  
Receptor Protein ◽  
Class Iii ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

scholarly journals In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase.

1975 ◽  
Vol 72 (7) ◽  
pp. 2506-2510 ◽  
Author(s):  
W. Zillig ◽  
H. Fujiki ◽  
W. Blum ◽  
D. Janekovic ◽  
M. Schweiger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Rna Polymerase ◽  
Bacteriophage T7

scholarly journals Additional Determinants within Escherichia coli FNR Activating Region 1 and RNA Polymerase α Subunit Required for Transcription Activation

2005 ◽  
Vol 187 (5) ◽  
pp. 1724-1731 ◽  
Author(s):  
K. Derek Weber ◽  
Owen D. Vincent ◽  
Patricia J. Kiley
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Class Ii ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Side Chains ◽  
Alanine Scanning Mutagenesis ◽  
Α Subunit

ABSTRACT The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(−61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the α subunit (αCTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that αCTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of αCTD required for FNR activation is more extensive than previously observed.

scholarly journals Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

2008 ◽  
Vol 190 (10) ◽  
pp. 3434-3443 ◽  
Author(s):  
Umender K. Sharma ◽  
Dipankar Chatterji
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Sigma Factors ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

Sign in / Sign up

Export Citation Format

Share Document