scholarly journals Conformational properties of Bacillus subtilis RNA polymerase σA factor during transcription initiation

1993 ◽  
Vol 294 (1) ◽  
pp. 43-47 ◽  
Author(s):  
B Y Chang ◽  
R H Doi
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Trypsin Digestion ◽  
Tryptic Fragment ◽  
The Core ◽  
Core Enzyme ◽  
Conformational Properties ◽  
Promoter Dna ◽  
Rna Polymerase Holoenzyme ◽  
Dna Complex

By the use of a partial proteolysis method and Western-blot analysis, the conformational properties of Bacillus subtilis sigma A factor in the transcription initiation stage were studied. From a comparison of the trypsin-digestion patterns of free sigma A and of sigma A associated with core enzyme, it was found that the production of 45 kDa sigma A tryptic-derived fragment was enhanced when sigma A was associated with the core enzyme. More importantly, a 40 kDa sigma A tryptic-derived fragment was found exclusively in this associated state. Based on the change of the digestion kinetics when producing the 45 kDa tryptic fragment and the generation of this new 40 kDa tryptic fragment from sigma A, it was apparent that a conformation change of sigma A occurred during the association of sigma A with the core enzyme. Also, similar patterns were found for the sigma A present in the holoenzyme-promoter DNA complex. These findings suggest that no further distinctive conformational change of sigma A occurs at the step of RNA polymerase holoenzyme and promoter DNA complex formation. Trypsin-digestion patterns of sigma A in different RNA polymerase holoenzyme and promoter DNA complexes were also studied. The presence of similar trypsin digestion-patterns of sigma A in those complexes strongly supports the idea that a similar sigma A conformation is used in the recognition of different sigma A-type promoters and the formation of different open complexes.

Investigations of the modular structure of bacterial promoters

2006 ◽  
Vol 73 ◽  
pp. 1-10 ◽  
Author(s):  
Nora S. Miroslavova ◽  
Stephen J.W. Busby
Keyword(s):  
Rna Polymerase ◽  
Dna Sequence ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Modular Structure ◽  
Bacterial Rna ◽  
Sequence Elements ◽  
Promoter Dna ◽  
Transcript Initiation ◽  
Rna Polymerase Holoenzyme

Bacterial RNA polymerase holoenzyme carries different determinants that contact different promoter DNA sequence elements. These contacts are essential for the recognition of promoters prior to transcript initiation. Here, we have investigated how active promoters can be built from different combinations of elements. Our results show that the contribution of different contacts to promoter activity is critically dependent on the overall promoter context, and that certain combinations of contacts can hinder transcription initiation.

scholarly journals Mutant Forms of Salmonella typhimuriumς54 Defective in Transcription Initiation but Not Promoter Binding Activity

1999 ◽  
Vol 181 (11) ◽  
pp. 3351-3357 ◽  
Author(s):  
Mary T. Kelly ◽  
Timothy R. Hoover
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Atp Hydrolysis ◽  
Random Mutagenesis ◽  
Binding Activity ◽  
Mutant Proteins ◽  
Promoter Dna ◽  
Mutant Forms ◽  
Rna Polymerase Holoenzyme

ABSTRACT Transcription initiation with ς54-RNA polymerase holoenzyme (ς54-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. ς54’s binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium ς54 that were deficient in transcription initiation but still directed ς54-holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for ς54-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of ς54 at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.

scholarly journals Crl Facilitates RNA Polymerase Holoenzyme Formation

2006 ◽  
Vol 188 (22) ◽  
pp. 7966-7970 ◽  
Author(s):  
Tamas Gaal ◽  
Mark J. Mandel ◽  
Thomas J. Silhavy ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Mechanism Of Action ◽  
Sigma Factor ◽  
Transcriptional Coactivator ◽  
Transcription System ◽  
The Core ◽  
Core Enzyme ◽  
Rna Polymerase Holoenzyme

ABSTRACT The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor σS. In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the EσS RNA polymerase holoenzyme but also by Eσ70 and Eσ32. Crl increased transcription dramatically but only when the σ concentration was low and when Crl was added to σ prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

scholarly journals Direct binding of TFEα opens DNA binding cleft of RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung-Hoon Jun ◽  
Jaekyung Hyun ◽  
Jeong Seok Cha ◽  
Hoyoung Kim ◽  
Michael S. Bartlett ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Transcription Bubble ◽  
Cryo Electron Microscopy ◽  
Direct Binding ◽  
Binding Cleft ◽  
Promoter Dna ◽  
Winged Helix Domain

AbstractOpening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

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