scholarly journals RNA polymerase clamp movement aids dissociation from DNA but is not required for RNA release at intrinsic terminators

2018 ◽  
Author(s):  
Michael J Bellecourt ◽  
Ananya Ray-Soni ◽  
Alex Harwig ◽  
Rachel Anne Mooney ◽  
Robert Landick
Keyword(s):  
Nucleic Acid ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Elongation Rate ◽  
List Type ◽  
Stem Loop ◽  
Dna Release ◽  
Transcription Complexes ◽  
Rna And Dna

ABSTRACTIn bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2-3 nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well-defined. RNAP contains a clamp domain that closes around the nucleic-acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine-crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.HighlightsDisulfide bond crosslinks probe the role of the RNAP clamp domain in terminationRNA but not DNA can release at terminators when the RNAP clamp is closedRestricting RNAP clamp movement affects elongation rate more than termination rateInhibiting TL conformational flexibility impairs both RNA and DNA release

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 Characterization of T7 RNA Polymerase Transcription Complexes Assembled on Nucleic Acid Scaffolds

2002 ◽  
Vol 277 (49) ◽  
pp. 47035-47043 ◽  
Author(s):  
Dmitri Temiakov ◽  
Michael Anikin ◽  
William T. McAllister
Keyword(s):  
Nucleic Acid ◽  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Transcription Complexes

scholarly journals E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

2019 ◽  
Author(s):  
James Chen ◽  
Saumya Gopalkrishnan ◽  
Courtney Chiu ◽  
Albert Y. Chen ◽  
Elizabeth A. Campbell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
E Coli ◽  
Bacterial Proteins ◽  
Cryo Electron Microscopy ◽  
Promoter Complex ◽  
Promoter Dna ◽  
Induced Changes

AbstractTraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp. How TraR and its homologs regulate transcription is unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis of RNAP conformational heterogeneity revealed TraR-induced changes in RNAP dynamics. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

scholarly journals Structural basis of RNA polymerase inhibition by viral and host factors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Simona Pilotto ◽  
Thomas Fouqueau ◽  
Natalya Lukoyanova ◽  
Carol Sheppard ◽  
Soizick Lucas-Staat ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Environmental Changes ◽  
Initiation Factor ◽  
Structural Basis ◽  
Regulation Of Transcription ◽  
Domains Of Life ◽  
Sulfolobus Turreted Icosahedral Virus

AbstractRNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

scholarly journals RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures

1991 ◽  
Vol 11 (3) ◽  
pp. 1508-1522
Author(s):  
S C Linn ◽  
D S Luse
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Adenovirus Type ◽  
Dnase I ◽  
Transcription Complexes ◽  
Adenovirus Type 2

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.

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

2016 ◽  
Vol 113 (15) ◽  
pp. 4051-4056 ◽  
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.

scholarly journals Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes

2008 ◽  
Vol 284 (4) ◽  
pp. 1962-1970 ◽  
Author(s):  
Elena K. Davydova ◽  
Irene Kaganman ◽  
Krystyna M. Kazmierczak ◽  
Lucia B. Rothman-Denes
Keyword(s):  
Nucleic Acid ◽  
Rna Polymerase ◽  
Bacteriophage N4 ◽  
Transcription Complexes ◽  
Nucleic Acid Interactions ◽  
N4 Virion

scholarly journals Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Timothy P. Newing ◽  
Aaron J. Oakley ◽  
Michael Miller ◽  
Catherine J. Dawson ◽  
Simon H. J. Brown ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Active Site ◽  
Molecular Basis ◽  
Replication Machinery ◽  
Gram Positive Bacteria ◽  
Cryo Electron Microscopy ◽  
Large Conformational Change ◽  
Transcription Complexes ◽  
Primary Channel

AbstractIn bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

scholarly journals Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries

2009 ◽  
Vol 29 (11) ◽  
pp. 2925-2934 ◽  
Author(s):  
Shankarling Krishnamurthy ◽  
Mohamed A. Ghazy ◽  
Claire Moore ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Serine Phosphorylation ◽  
Initiation Factor ◽  
Prolyl Isomerase ◽  
Pol Ii ◽  
Proline Isomerization ◽  
Transcription Cycle

ABSTRACT The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a reiterated heptad sequence (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) that plays a key role in the transcription cycle, coordinating the exchange of transcription and RNA processing factors. The structure of the CTD is flexible and undergoes conformational changes in response to serine phosphorylation and proline isomerization. Here we report that the Ess1 peptidyl prolyl isomerase functionally interacts with the transcription initiation factor TFIIB and with the Ssu72 CTD phosphatase and Pta1 components of the CPF 3′-end processing complex. The ess1 A144T and ess1 H164R mutants, initially described by Hanes and coworkers (Yeast 5:55-72, 1989), accumulate the pSer5 phosphorylated form of Pol II; confer phosphate, galactose, and inositol auxotrophies; and fail to activate PHO5, GAL10, and INO1 reporter genes. These mutants are also defective for transcription termination, but in vitro experiments indicate that this defect is not caused by altering the processing efficiency of the cleavage/polyadenylation machinery. Consistent with a role in initiation and termination, Ess1 associates with the promoter and terminator regions of the PMA1 and PHO5 genes. We propose that Ess1 facilitates pSer5-Pro6 dephosphorylation by generating the CTD structural conformation recognized by the Ssu72 phosphatase and that pSer5 dephosphorylation affects both early and late stages of the transcription cycle.

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