A Single Molecule FRET Study of Formation of RNA Polymerase II Elongation Complex on Passivated Surface

2010 ◽  
Vol 57 (3B) ◽  
pp. 514-521 ◽  
Author(s):  
Yen-Chen Lin ◽  
Bo-Lin Lin ◽  
Tommy Setiawan ◽  
Chia-Chi Chang ◽  
Chi-Fu Yen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Elongation Complex ◽  
Single Molecule Fret

scholarly journals The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

2018 ◽  
Author(s):  
Diego Duchi ◽  
Abhishek Mazumder ◽  
Anssi M. Malinen ◽  
Richard H. Ebright ◽  
Achillefs N. Kapanidis
Keyword(s):  
Rna Polymerase ◽  
Active Center ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Stringent Response ◽  
Elongation Complex ◽  
Conformational States ◽  
Closed State ◽  
Single Molecule Fret ◽  
Promoter Dna

ABSTRACTRNA polymerase (RNAP) contains a mobile structural module, the “clamp,” that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including loading of promoter DNA into the RNAP active-center cleft, unwinding of promoter DNA, transcription elongation complex stability, transcription pausing, and transcription termination. Crystal structures and single-molecule FRET studies establish that the clamp can adopt open and closed conformational states; however, the occurrence, pathway, and kinetics of transitions between clamp states have been unclear. Using single-molecule FRET (smFRET) on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme exists in three distinct conformational states: the previously defined open state, the previously defined closed state, and a previously undefined partly closed state. smFRET time-traces show dynamic transitions between open, partly closed, and closed states on the 0.1-1 second time-scale. Similar analyses of transcription initiation complexes confirm that the RNAP clamp is closed in the catalytically competent transcription initiation complex and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, in contrast to in RNAP holoenzyme, the clamp does not interconvert between the closed state and other states. The stringent-response alarmone ppGpp selectively stabilizes the partly-closed-clamp state, inhibiting interconversion between the partly closed state and the open state. The methods of this report should allow elucidation of clamp conformation and dynamics during all phases of transcription.SIGNIFICANCE STATEMENTThe clamp forms a pincer of the RNA polymerase “crab-claw” structure, and adopts many conformations with poorly understood function and dynamics. By measuring distances within single surface-attached molecules, we observe directly the motions of the clamp and show that it adopts an open, a closed, and a partly closed state; the last state is stabilized by a sensor of bacterial starvation, linking the clamp conformation to the mechanisms used by bacteria to counteract stress. We also show that the clamp remains closed in many transcription steps, as well as in the presence of a specific antibiotic. Our approach can monitor clamp motions throughout transcription and offers insight on how antibiotics can stop pathogens by blocking their RNA polymerase movements.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

Allosteric transcription stimulation by RNA polymerase II super elongation complex

2021 ◽  
Author(s):  
Ying Chen ◽  
Seychelle M. Vos ◽  
Christian Dienemann ◽  
Momchil Ninov ◽  
Henning Urlaub ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Complex ◽  
Super Elongation Complex

scholarly journals Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex

1993 ◽  
Vol 21 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Gretchen A. Rice ◽  
Michael J. Chamberlin ◽  
Caroline M. Kane
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Complex ◽  
Template Dna

Structure of the complete elongation complex of RNA polymerase II with basal factors

Science ◽  
2017 ◽  
Vol 357 (6354) ◽  
pp. 921-924 ◽  
Author(s):  
Haruhiko Ehara ◽  
Takeshi Yokoyama ◽  
Hideki Shigematsu ◽  
Shigeyuki Yokoyama ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Complex

scholarly journals Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex

2009 ◽  
Vol 37 (17) ◽  
pp. 5803-5809 ◽  
Author(s):  
Joanna Andrecka ◽  
Barbara Treutlein ◽  
Maria Angeles Izquierdo Arcusa ◽  
Adam Muschielok ◽  
Robert Lewis ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Positioning System ◽  
Elongation Complex
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