Concerted Protein and Nucleic Acid Conformational Changes Observed Prior to Nucleotide Incorporation in a Bacterial RNA Polymerase: Raman Crystallographic Evidence

Biochemistry ◽  
2015 ◽  
Vol 54 (34) ◽  
pp. 5297-5305 ◽  
Author(s):  
Ioanna H. Antonopoulos ◽  
Brittany A. Warner ◽  
Paul R. Carey
Keyword(s):  
Nucleic Acid ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Nucleotide Incorporation ◽  
Bacterial Rna

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

scholarly journals The extent of B-form structure in a 6S RNA variant that mimics DNA to bind to the Escherichia coli RNA Polymerase

2018 ◽  
Author(s):  
Nilesh K. Banavali
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Structural Analysis ◽  
Rna Polymerase ◽  
Rna Structure ◽  
Recent Article ◽  
Single Strand ◽  
Bacterial Rna ◽  
6S Rna

AbstractIn a recent article by Darst and coworkers, it was found that a non-coding 6S RNA variant regulates a bacterial RNA polymerase by mimicking B-Form DNA, and a few different nucleic acid duplex parameters were analyzed to understand the extent of B-form RNA structure. In this manuscript, a different structural analysis based on conformational distance from canonical A-form and B-form single-strand structures is presented. This analysis addresses the occurrence and extent of both local and global B-form structure in the published 6S RNA variant model.

scholarly journals Engagement of Arginine Finger to ATP Triggers Large Conformational Changes in NtrC1 AAA+ ATPase for Remodeling Bacterial RNA Polymerase

Structure ◽  
2010 ◽  
Vol 18 (11) ◽  
pp. 1420-1430 ◽  
Author(s):  
Baoyu Chen ◽  
Tatyana A. Sysoeva ◽  
Saikat Chowdhury ◽  
Liang Guo ◽  
Sacha De Carlo ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Aaa Atpase ◽  
Bacterial Rna ◽  
Arginine Finger

scholarly journals RNA polymerase clamp movement aids dissociation from DNA but is not required for RNA release at intrinsic terminators

2018 ◽  
Author(s):  
Michael J Bellecourt ◽  
Ananya Ray-Soni ◽  
Alex Harwig ◽  
Rachel Anne Mooney ◽  
Robert Landick
Keyword(s):  
Nucleic Acid ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Elongation Rate ◽  
List Type ◽  
Stem Loop ◽  
Dna Release ◽  
Transcription Complexes ◽  
Rna And Dna

ABSTRACTIn bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2-3 nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well-defined. RNAP contains a clamp domain that closes around the nucleic-acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine-crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.HighlightsDisulfide bond crosslinks probe the role of the RNAP clamp domain in terminationRNA but not DNA can release at terminators when the RNAP clamp is closedRestricting RNAP clamp movement affects elongation rate more than termination rateInhibiting TL conformational flexibility impairs both RNA and DNA release

scholarly journals Ureidothiophene inhibits interaction of bacterial RNA polymerase with –10 promotor element

2020 ◽  
Vol 48 (14) ◽  
pp. 7914-7923
Author(s):  
John Harbottle ◽  
Nikolay Zenkin
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Early Stage ◽  
Chemical Compounds ◽  
Initiation Factor ◽  
Promoter Element ◽  
Bacterial Transcription ◽  
Bacterial Rna ◽  
Novel Target

Abstract Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter –10 element, while not affecting interactions with –35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of –10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with –10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.

scholarly journals Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

2017 ◽  
Vol 114 (44) ◽  
pp. E9233-E9242 ◽  
Author(s):  
Ananya Ray-Soni ◽  
Rachel A. Mooney ◽  
Robert Landick
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Dna Sequences ◽  
Time Window ◽  
Kinetic Assay ◽  
Termination Rate ◽  
Trigger Loop ◽  
Bacterial Rna ◽  
Nucleotide Addition ◽  
Addition Rate

In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA−DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991–1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.

scholarly journals Time-Resolved Raman and Polyacrylamide Gel Electrophoresis Observations of Nucleotide Incorporation and Misincorporation in RNA within a Bacterial RNA Polymerase Crystal

Biochemistry ◽  
2015 ◽  
Vol 54 (3) ◽  
pp. 652-665 ◽  
Author(s):  
Ioanna H. Antonopoulos ◽  
Yuko Murayama ◽  
Brittany A. Warner ◽  
Shun-ichi Sekine ◽  
Shigeyuki Yokoyama ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Time Resolved ◽  
Nucleotide Incorporation ◽  
Bacterial Rna
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