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 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 The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo A. Chen ◽  
Yong-Heng Huang ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Slow Growth ◽  
Coiled Coil ◽  
Main Channel ◽  
Dependent Manner ◽  
Secondary Channel

AbstractCellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

An energy charge sensor for balancing RNA polymerase recycling and hibernation

2020 ◽  
Author(s):  
Markus Wahl ◽  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo Chen ◽  
Yong-Heng Huang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Energy Levels ◽  
Energy Charge ◽  
Coiled Coil ◽  
Main Channel ◽  
Rna Polymerase I ◽  
Secondary Channel ◽  
Polymerase I

Abstract Cellular RNA polymerases can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, or enter dormancy. How RNA polymerase recycling into active states is achieved and balanced with quiescence remains elusive. We structurally analyzed Bacillus subtilis RNA polymerase bound to the NTPase HelD. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNA polymerase secondary channel, dismantling the active site and displacing RNA; a unique helical protrusion inserts into the main channel, prying β and β’ subunits apart and dislodging DNA, aided by the δ subunit. HelD release depends on ATP, and a dimeric structure resembling hibernating RNA polymerase I suggests that HelD can induce dormancy at low energy levels. Our results reveal an ingenious mechanism by which active RNA polymerase pools are adjusted in response to the nutritional state.

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.

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