scholarly journals Using Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during σ70 Binding

2002 ◽  
Vol 184 (10) ◽  
pp. 2634-2641 ◽  
Author(s):  
Larry C. Anthony ◽  
Alan A. Dombkowski ◽  
Richard R. Burgess
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Disulfide Bond ◽  
Conformational Changes ◽  
Coiled Coil ◽  
Mrna Synthesis ◽  
Promoter Sequences ◽  
Coiled Coil Region

ABSTRACT RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the β′ subunit (β′260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with σ70 both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ70. In this work, we engineered a disulfide bond within β′240-309 that locks the β′ coiled-coil region in the coiled-coil conformation, and we show that this “locked” peptide is able to bind to σ70. We also show that the locked coiled-coil is capable of inducing a conformational change within σ70 that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ70 or upon recognition of the −10 nontemplate strand of DNA.

scholarly journals Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

2009 ◽  
Vol 191 (24) ◽  
pp. 7614-7619 ◽  
Author(s):  
Rong Shi ◽  
Magda Villarroya ◽  
Rafael Ruiz-Partida ◽  
Yunge Li ◽  
Ariane Proteau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Function Analysis ◽  
Trna Modification ◽  
Conserved Residues ◽  
Limited Trypsinolysis ◽  
Fad Binding ◽  
Trna Binding

ABSTRACT The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

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 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 Cloning and Characterization of SCHIP-1, a Novel Protein Interacting Specifically with Spliced Isoforms and Naturally Occurring Mutant NF2 Proteins

2000 ◽  
Vol 20 (5) ◽  
pp. 1699-1712 ◽  
Author(s):  
Laurence Goutebroze ◽  
Estelle Brault ◽  
Christian Muchardt ◽  
Jacques Camonis ◽  
Gilles Thomas
Keyword(s):  
Tumor Suppressor ◽  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Coiled Coil ◽  
Neurofibromatosis Type ◽  
Immunofluorescent Staining ◽  
Naturally Occurring ◽  
Cellular Context

ABSTRACT The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved in NF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members, implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as a tumor suppressor is unknown. In attempt to shed light on schwannomin function, we have identified a novel coiled-coil protein, SCHIP-1, that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein is homologous to human FEZ proteins and the relatedCaenorhabditis elegans gene product UNC-76. Immunofluorescent staining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmic membrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can be observed only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and 3, but not with the schwannomin isoform exhibiting growth-suppressive activity. Our observations suggest that SCHIP-1 interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslational modifications, alternative splicing, or mutations.

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.

Demonstration that the TyrR Protein and RNA Polymerase Complex Formed at the Divergent P3 Promoter Inhibits Binding of RNA Polymerase to the Major Promoter, P1, of the aroP Gene ofEscherichia coli

1998 ◽  
Vol 180 (20) ◽  
pp. 5466-5472 ◽  
Author(s):  
Peixiang Wang ◽  
Ji Yang ◽  
Akira Ishihama ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Region ◽  
Ofescherichia Coli ◽  
Mrna Synthesis ◽  
Polymerase Complex ◽  
Defective Mutant ◽  
Dna Fragment ◽  
Region Ii

ABSTRACT In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213–4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

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.

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