Studies in vivo on Escherichia coli RNA polymerase mutants altered in the stringent response

1988 ◽  
Vol 213 (2-3) ◽  
pp. 379-387 ◽  
Author(s):  
E. Baracchini ◽  
R. Glass ◽  
H. Bremer
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stringent Response ◽  
Polymerase Mutants

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.

scholarly journals Inorganic Polyphosphate Accumulation inEscherichia coliIs Regulated by DksA but Not by (p)ppGpp

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Michael J. Gray
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Elongation Factor ◽  
Stringent Response ◽  
Inorganic Polyphosphate ◽  
Content Type ◽  
Polyphosphate Accumulation ◽  
Pathogenic Microbes ◽  
Polyp Accumulation

ABSTRACTProduction of inorganic polyphosphate (polyP) by bacteria is triggered by a variety of different stress conditions. polyP is required for stress survival and virulence in diverse pathogenic microbes. Previous studies have hypothesized a model for regulation of polyP synthesis in which production of the stringent-response second messenger (p)ppGpp directly stimulates polyP accumulation. In this work, I have now shown that this model is incorrect, and (p)ppGpp is not required for polyP synthesis inEscherichia coli. However, stringent mutations of RNA polymerase that frequently arise spontaneously in strains defective in (p)ppGpp synthesis and null mutations of the stringent-response-associated transcription factor DksA both strongly inhibit polyP accumulation. The loss of polyP synthesis in a mutant lacking DksA was reversed by deletion of the transcription elongation factor GreA, suggesting that competition between these proteins for binding to the secondary channel of RNA polymerase plays an important role in controlling polyP activation. These results provide new insights into the poorly understood regulation of polyP synthesis in bacteria and indicate that the relationship between polyP and the stringent response is more complex than previously suspected.IMPORTANCEProduction of polyP in bacteria is required for virulence and stress response, but little is known about how bacteria regulate polyP levels in response to changes in their environments. Understanding this regulation is important for understanding how pathogenic microbes resist killing by disinfectants, antibiotics, and the immune system. In this work, I have clarified the connections between polyP regulation and the stringent response to starvation stress inEscherichia coliand demonstrated an important and previously unknown role for the transcription factor DksA in controlling polyP levels.

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 In Vivo Effect of NusB and NusG on rRNA Transcription Antitermination

2004 ◽  
Vol 186 (5) ◽  
pp. 1304-1310 ◽  
Author(s):  
Martha Torres ◽  
Joan-Miquel Balada ◽  
Malcolm Zellars ◽  
Craig Squires ◽  
Catherine L. Squires
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Protein Complex ◽  
Test System ◽  
Wild Type ◽  
Rrna Transcription ◽  
Transcription Antitermination ◽  
Gene Mrna

ABSTRACT Similarities between lambda and rRNA transcription antitermination have led to suggestions that they involve the same Nus factors. However, direct in vivo confirmation that rRNA antitermination requires all of the lambda Nus factors is lacking. We have therefore analyzed the in vivo role of NusB and NusG in rRNA transcription antitermination and have established that both are essential for it. We used a plasmid test system in which reporter gene mRNA was measured to monitor rRNA antiterminator-dependent bypass of a Rho-dependent terminator. A comparison of terminator read-through in a wild-type Escherichia coli strain and that in a nusB::IS10 mutant strain determined the requirement for NusB. In the absence of NusB, antiterminator-dependent terminator read-through was not detected, showing that NusB is necessary for rRNA transcription antitermination. The requirement for NusG was determined by comparing rRNA antiterminator-dependent terminator read-through in a strain overexpressing NusG with that in a strain depleted of NusG. In NusG-depleted cells, termination levels were unchanged in the presence or absence of the antiterminator, demonstrating that NusG, like NusB, is necessary for rRNA transcription antitermination. These results imply that NusB and NusG are likely to be part of an RNA-protein complex formed with RNA polymerase during transcription of the rRNA antiterminator sequences that is required for rRNA antiterminator-dependent terminator read-through.

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.

Sign in / Sign up

Export Citation Format

Share Document