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

2007 ◽  
Vol 27 (3) ◽  
pp. 406-419 ◽  
Author(s):  
Innokenti Toulokhonov ◽  
Jinwei Zhang ◽  
Murali Palangat ◽  
Robert Landick
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Trigger Loop ◽  
Transcriptional Pausing

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 Regulation of transcriptional pausing through the secondary channel of RNA polymerase

2016 ◽  
Vol 113 (31) ◽  
pp. 8699-8704 ◽  
Author(s):  
Daria Esyunina ◽  
Aleksei Agapov ◽  
Andrey Kulbachinskiy
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Active Site ◽  
Rna Structure ◽  
Deinococcus Radiodurans ◽  
Genetic Regulation ◽  
Secondary Channel ◽  
Trigger Loop ◽  
Transcriptional Pausing ◽  
External Stimuli

Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stress-resistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg2+ or Mn2+) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.

scholarly journals Role of the RNA polymerase trigger loop in catalysis and pausing

2009 ◽  
Vol 17 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Jinwei Zhang ◽  
Murali Palangat ◽  
Robert Landick
Keyword(s):  
Rna Polymerase ◽  
Trigger Loop

scholarly journals Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

2015 ◽  
Vol 112 (5) ◽  
pp. E410-E419 ◽  
Author(s):  
Celine Walmacq ◽  
Lanfeng Wang ◽  
Jenny Chong ◽  
Kathleen Scibelli ◽  
Lucyna Lubkowska ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Dna Lesions ◽  
Abasic Site ◽  
Pol Ii ◽  
Dna Lesion ◽  
Trigger Loop ◽  
Pyrimidine Dimers ◽  
Bulky Dna Lesions

In human cells, the oxidative DNA lesion 8,5′-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5′) next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5′-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

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.

scholarly journals High-resolution phenotypic landscape of the RNA Polymerase II trigger loop

2016 ◽  
Author(s):  
Chenxi Qiu ◽  
Olivia C. Erinne ◽  
Jui Dave ◽  
Ping Cui ◽  
Huiyan Jin ◽  
...  
Keyword(s):  
High Resolution ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Genetic Interactions ◽  
Substrate Selection ◽  
Phenotypic Data ◽  
Trigger Loop ◽  
Allele Specific ◽  
Individual Residue

The active site of multicellular RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three major mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.

scholarly journals The Rpb4 Subunit of RNA Polymerase II Contributes to Cotranscriptional Recruitment of 3′ Processing Factors

2008 ◽  
Vol 28 (6) ◽  
pp. 1883-1891 ◽  
Author(s):  
Vanessa M. Runner ◽  
Vladimir Podolny ◽  
Stephen Buratowski
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Chromatin Immunoprecipitation ◽  
The Core ◽  
Catalytic Active Site ◽  
Cross Links ◽  
Processing Factors

ABSTRACT The RNA polymerase II enzyme from the yeast Saccharomyces cerevisiae is a complex of 12 subunits, Rpb1 to Rpb12. Crystal structures of the full complex show that the polymerase consists of two separable components, a 10-subunit core including the catalytic active site and a heterodimer of the Rpb4 and Rpb7 subunits. To characterize the role of the Rpb4/7 heterodimer during transcription in vivo, chromatin immunoprecipitation was used to examine an rpb4Δ strain for effects on the behavior of the core polymerase as well as recruitment of other protein factors involved in transcription. Rpb4/7 cross-links throughout transcribed regions. Loss of Rpb4 results in a reduction of RNA polymerase II levels near 3′ ends of multiple mRNA genes as well as a decreased association of 3′-end processing factors. Furthermore, loss of Rpb4 results in altered polyadenylation site usage at the RNA14 gene. Together, these results indicate that Rpb4 contributes to proper cotranscriptional 3′-end processing in vivo.

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