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

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.

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Computer-aided structural and molecular insights into the mechanisms by which Pseudouridimycin (PUM) disrupts cleft extension in bacterial RNA polymerase to block DNA entry and exit

Letters in Drug Design & Discovery ◽

10.2174/1570180817999201123165144 ◽

2020 ◽

Vol 17 ◽

Author(s):

Ali H. Rabbad ◽

Fisayo A. Olotu ◽

Mahmoud E. Soliman

Keyword(s):

Rna Polymerase ◽

Bacterial Infections ◽

Entry And Exit ◽

Dynamic Binding ◽

Binding Behavior ◽

Bacterial Rna ◽

Nucleotide Addition ◽

Switch Regions ◽

Cleft Extension ◽

Key Residues

Background: The ability of Pseudouridimycin (PUM) to occupy the nucleotide addition site of bacterial RNA Polymerase (RNAP) underlies its inhibitory potency as previously reported. PUM has gained high research interest as a broad-spectrum nucleoside analog that has demonstrated exciting potentials in treating drug-resistant bacterial infections. Objective: Herein, we identified, for the first time, a novel complementary mechanism by which PUM elicits its inhibitory effects on bacterial RNAP. Methods: The dynamic binding behavior of PUM to bacterial RNAP was studied using various dynamic analyses approaches. Results and Discussion: Findings revealed that in addition to occupying the nucleotide addition site, PUM also interrupts the unimpeded entry and exit of DNA by reducing the mechanistic extension of the RNAP cleft and perturbing the primary conformations of the switch regions. Moreover, PUM binding reduced the distances between key residues in the β and β’ subunits that extend to accommodate the DNA. Conclusion: This study’s findings present structural insights that would contribute to the structure-based design of potent and selective PUM inhibitors.

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THEORETICAL INVESTIGATIONS ON ELUCIDATING FUNDAMENTAL MECHANISMS OF CATALYSIS AND DYNAMICS INVOLVED IN TRANSCRIPTION BY RNA POLYMERASE

Journal of Theoretical and Computational Chemistry ◽

10.1142/s0219633613410058 ◽

2013 ◽

Vol 12 (08) ◽

pp. 1341005 ◽

Cited By ~ 4

Author(s):

FÁTIMA PARDO-AVILA ◽

LIN-TAI DA ◽

YING WANG ◽

XUHUI HUANG

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Normal Mode Analysis ◽

Md Simulations ◽

Mode Analysis ◽

Transcription Process ◽

Nucleotide Addition ◽

Theoretical Investigations ◽

State Models ◽

Elongation Process

RNA polymerase is the enzyme that synthesizes RNA during the transcription process. To understand its mechanism, structural studies have provided us pictures of the series of steps necessary to add a new nucleotide to the nascent RNA chain, the steps altogether known as the nucleotide addition cycle (NAC). However, these static snapshots do not provide dynamic information of these processes involved in NAC, such as the conformational changes of the protein and the atomistic details of the catalysis. Computational studies have made efforts to fill these knowledge gaps. In this review, we provide examples of different computational approaches that have improved our understanding of the transcription elongation process for RNA polymerase, such as normal mode analysis, molecular dynamic (MD) simulations, Markov state models (MSMs). We also point out some unsolved questions that could be addressed using computational tools in the future.

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The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

Journal of Bacteriology ◽

10.1128/jb.00583-18 ◽

2018 ◽

Vol 201 (4) ◽

Cited By ~ 1

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.

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Closing and opening of the RNA polymerase trigger loop

10.1101/817080 ◽

2019 ◽

Cited By ~ 1

Author(s):

Abhishek Mazumder ◽

Miaoxin Lin ◽

Achillefs N. Kapanidis ◽

Richard H. Ebright

Keyword(s):

Rna Polymerase ◽

Fluorescent Probe ◽

Active Center ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Single Molecule Fluorescence ◽

Structural Element ◽

Trigger Loop ◽

Nucleotide Addition

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an “unfolded”/“open” state that allows an NTP substrate to enter the active center and a “folded”/“closed” state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

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

Structure ◽

10.1016/j.str.2010.08.018 ◽

2010 ◽

Vol 18 (11) ◽

pp. 1420-1430 ◽

Cited By ~ 38

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

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Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421067112 ◽

2014 ◽

Vol 112 (3) ◽

pp. 743-748 ◽

Cited By ~ 31

Author(s):

Yara X. Mejia ◽

Evgeny Nudler ◽

Carlos Bustamante

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Single Molecule ◽

Conformational Changes ◽

Elongation Rate ◽

Nucleoside Triphosphate ◽

Transcription Rate ◽

Nucleotide Analogs ◽

Catalytic Center ◽

Trigger Loop

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP’s pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa591 ◽

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.

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Transcription inhibition by the depsipeptide antibiotic salinamide A

eLife ◽

10.7554/elife.02451 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 52

Author(s):

David Degen ◽

Yu Feng ◽

Yu Zhang ◽

Katherine Y Ebright ◽

Yon W Ebright ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Transcription Initiation ◽

Rna Synthesis ◽

Antibacterial Drug ◽

Transcription Inhibition ◽

Bacterial Rna ◽

Cellular Target ◽

Nucleotide Addition ◽

And Function

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.

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Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520555113 ◽

2016 ◽

Vol 113 (15) ◽

pp. 4051-4056 ◽

Cited By ~ 54

Author(s):

Bin Liu ◽

Yuhong Zuo ◽

Thomas A. Steitz

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Active Site ◽

Transcription Initiation ◽

De Novo ◽

Addition Reaction ◽

Specific Interactions ◽

Promoter Recognition ◽

Nucleotide Addition ◽

Promoter Dna

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

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Obligate movements of an active site–linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2101805118 ◽

2021 ◽

Vol 118 (36) ◽

pp. e2101805118

Author(s):

Yu Bao ◽

Robert Landick

Keyword(s):

Rna Polymerase ◽

Channel Trafficking ◽

Positional Bias ◽

Trigger Loop ◽

Nucleotide Addition ◽

Helical Hairpin ◽

Transcript Elongation ◽

Transcriptional Pausing ◽

Active Transcription ◽

Transcription Complexes

The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H2O2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition.

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