scholarly journals CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition

2015 ◽  
Vol 112 (31) ◽  
pp. E4178-E4187 ◽  
Author(s):  
Brian Bae ◽  
Dhananjaya Nayak ◽  
Ananya Ray ◽  
Arkady Mustaev ◽  
Robert Landick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Terminal Portion ◽  
Biochemical Tests ◽  
Catalytic Center ◽  
Mediated Effects ◽  
Polymerase Inhibitors ◽  
Trigger Loop ◽  
Nucleotide Addition ◽  
Hydrolysis Of

RNA polymerase inhibitors like the CBR class that target the enzyme’s complex catalytic center are attractive leads for new antimicrobials. Catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg2+ ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. We report crystal structures of CBR inhibitor/Escherichia coli RNA polymerase complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3′-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.

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

2014 ◽  
Vol 112 (3) ◽  
pp. 743-748 ◽  
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.

scholarly journals Closing and opening of the RNA polymerase trigger loop

2019 ◽  
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.

Affinity labeling of a cysteine at or near the catalytic center of Escherichia coli B DNA-dependent RNA polymerase

1980 ◽  
Vol 612 (1) ◽  
pp. 286-294 ◽  
Author(s):  
Jeffrey A. Miller ◽  
Gary F. Serio ◽  
John L. Bear ◽  
Robert A. Howard ◽  
Aubrey P. Kimball
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Affinity Labeling ◽  
Catalytic Center ◽  
Escherichia Coli B

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

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.

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

2021 ◽  
Author(s):  
Yu Bao ◽  
Robert Landick
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Reporter System ◽  
Channel Trafficking ◽  
Trigger Loop ◽  
Nucleotide Addition ◽  
Helical Hairpin ◽  
Transcript Elongation ◽  
Active Transcription ◽  
Transcription Complexes

ABSTRACTThe 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 2–5 surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-aa, 2-SBHM E. coli insertion, called SI3, occupies different locations in halted, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here we report 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 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 the TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition.SIGNIFICANCERNA synthesis by cellular RNA polymerases depends on an active-site component called the trigger loop that oscillates between an unstructured loop that admits NTP substrates and a helical hairpin that positions the NTP in every round of nucleotide addition. In most bacteria, the trigger loop contains a large, surface-exposed insertion module that occupies different positions in halted transcription complexes but whose function during active transcription is unknown. By developing and using a novel disulfide reporter system, we find the insertion module also must alternate between in and out positions for every nucleotide addition, must swivel to a paused position to support regulation, and, in enterobacteria, evolved a “Phe pocket” that captures a key phenylalanine in the out and swivel positions.

5'-O-diphosphorylphosphonylmethylribonucleosides - A new group of DNA-dependent RNA polymerase inhibitors

1983 ◽  
Vol 48 (5) ◽  
pp. 1352-1357 ◽  
Author(s):  
Květa Horská ◽  
Ivan Rosenberg ◽  
Antonín Holý ◽  
Karel Šebesta
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Purine Derivatives ◽  
Pyrimidine Derivatives ◽  
Polymerase Inhibitors ◽  
Enzyme Substrates ◽  
Inhibition Constants

The effect of 5'-O-diphosphorylphosphonylmethylribonucleosides I on the transcription reaction catalyzed by DNA-dependent RNA polymerase from Escherichia coli was studied. These analogues of ribonucleoside 5'-triphosphates are not enzyme substrates but inhibit the transcription reaction, competing specifically with the natural substrates. According to the inhibition constants, the pyrimidine derivatives Ia,b are more potent inhibitors than the purine derivatives Ic,d.

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 Closing and opening of the RNA polymerase trigger loop

2020 ◽  
Vol 117 (27) ◽  
pp. 15642-15649 ◽  
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.

scholarly journals Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

2020 ◽  
Vol 295 (28) ◽  
pp. 9583-9595
Author(s):  
Aleksei Agapov ◽  
Artem Ignatov ◽  
Matti Turtola ◽  
Georgiy Belogurov ◽  
Daria Esyunina ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Stress Responses ◽  
Active Site ◽  
Rna Synthesis ◽  
Conformational Dynamics ◽  
Dna Lesions ◽  
Trigger Loop ◽  
Nucleotide Addition

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

scholarly journals DNA Bending and Wrapping around RNA Polymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms

1999 ◽  
Vol 63 (2) ◽  
pp. 457-478 ◽  
Author(s):  
Benoit Coulombe ◽  
Zachary F. Burton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Dna Bending ◽  
Tata Binding Protein ◽  
Transcriptional Activators ◽  
Catalytic Center ◽  
Transcription Bubble ◽  
Rna Polymerase Ii Transcription

SUMMARY A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription.

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