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

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.

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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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Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1806 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1806-1814 ◽

Cited By ~ 11

Author(s):

H S Sullivan ◽

L S Young ◽

C N White ◽

K U Sprague

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Silk Gland ◽

Flanking Sequence ◽

Transcription Machinery ◽

Polymerase Iii ◽

Active Transcription ◽

The Difference ◽

Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

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Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1806-1814.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1806-1814

Author(s):

H S Sullivan ◽

L S Young ◽

C N White ◽

K U Sprague

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Silk Gland ◽

Flanking Sequence ◽

Transcription Machinery ◽

Polymerase Iii ◽

Active Transcription ◽

The Difference ◽

Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

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Role of the RNA polymerase trigger loop in transcript elongation, cleavage, and pausing

The FASEB Journal ◽

10.1096/fasebj.23.1_supplement.430.2 ◽

2009 ◽

Vol 23 (S1) ◽

Author(s):

Robert Landick

Keyword(s):

Rna Polymerase ◽

Trigger Loop ◽

Transcript Elongation

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A Central Role of the RNA Polymerase Trigger Loop in Active-Site Rearrangement during Transcriptional Pausing

Molecular Cell ◽

10.1016/j.molcel.2007.06.008 ◽

2007 ◽

Vol 27 (3) ◽

pp. 406-419 ◽

Cited By ~ 160

Author(s):

Innokenti Toulokhonov ◽

Jinwei Zhang ◽

Murali Palangat ◽

Robert Landick

Keyword(s):

Rna Polymerase ◽

Active Site ◽

Trigger Loop ◽

Transcriptional Pausing

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706247114 ◽

2017 ◽

Vol 114 (44) ◽

pp. E9233-E9242 ◽

Cited By ~ 8

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.

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Closing and opening of the RNA polymerase trigger loop

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920427117 ◽

2020 ◽

Vol 117 (27) ◽

pp. 15642-15649 ◽

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011844 ◽

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502368112 ◽

2015 ◽

Vol 112 (31) ◽

pp. E4178-E4187 ◽

Cited By ~ 26

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.

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