scholarly journals Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA

2019 ◽  
Author(s):  
Wooyoung Kang ◽  
Kook Sun Ha ◽  
Heesoo Uhm ◽  
Kyuhyong Park ◽  
Ja Yil Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Sigma Factor ◽  
Single Molecule Fluorescence ◽  
Fluorescence Study ◽  
Transcription Reinitiation ◽  
Dna Template ◽  
Rna Transcript

(Abstract)Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from DNA template in bacterial intrinsic termination of transcription much more often than concurrent dissociation. As termination is defined by release of product RNA from transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as ‘recycling.’ During the recycling stage, RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and these RNAPs can initiate transcription again at nearby promoters in case of retaining a sigma factor. The efficiency of this event, termed here as ‘reinitiation,’ increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on DNA template for reinitiation most times.

scholarly journals Closing and opening of the RNA polymerase trigger loop

2019 ◽  
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.

scholarly journals Fast Biosynthesis of GFP Molecules - A Single Molecule Fluorescence Study

2010 ◽  
Vol 98 (3) ◽  
pp. 259a-260a
Author(s):  
Georg Büldt ◽  
Alexandros Katranidis ◽  
Ramona Schlesinger ◽  
Knud H. Nierhaus ◽  
Ingo Gregor ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Fluorescence Study

scholarly journals Single-Molecule Fluorescence Study of RNA Recognition by Viral RNAi Suppressors

2017 ◽  
Vol 112 (3) ◽  
pp. 151a
Author(s):  
Mohamed Fareh ◽  
Jasper van Lopik ◽  
Iason Katechis ◽  
Ronald van Rij ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Rna Recognition ◽  
Single Molecule Fluorescence ◽  
Fluorescence Study

scholarly journals Single Molecule Fluorescence Study of G-Quadruplex Sensor

2015 ◽  
Vol 108 (2) ◽  
pp. 164a
Author(s):  
Yingya Li ◽  
Xiao Fan ◽  
Yanyan Li ◽  
Haitao Li
Keyword(s):  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Fluorescence Study ◽  
G Quadruplex

scholarly journals Influence of DNA Template Choice on Transcription and Inhibition of Escherichia coli RNA Polymerase

2012 ◽  
Vol 56 (8) ◽  
pp. 4536-4539 ◽  
Author(s):  
Joerg Haupenthal ◽  
Kristina Hüsecken ◽  
Matthias Negri ◽  
Christine K. Maurer ◽  
Rolf W. Hartmann
Keyword(s):  
Escherichia Coli ◽  
Drug Discovery ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Inhibitory Potency ◽  
Experimental Conditions ◽  
Early Stages ◽  
Bacterial Rna ◽  
Transcription Efficiency ◽  
Dna Template

ABSTRACTIn recent decades, quantitative transcription assays using bacterial RNA polymerase (RNAP) have been performed under widely diverse experimental conditions. We demonstrate that the template choice can influence the inhibitory potency of RNAP inhibitors. Furthermore, we illustrate that the sigma factor (σ70) surprisingly increases the transcription efficiency of templates with nonphysiological nonprokaryotic promoters. Our results might be a useful guideline in the early stages of using RNAP for drug discovery.

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