Single molecule fluorescence study of DNA helicase activity

2000 ◽  
Author(s):  
T. Ha ◽  
H.P. Babcock ◽  
W. Cheng ◽  
T.M. Lohman ◽  
S. Chu
Keyword(s):  
Single Molecule ◽  
Dna Helicase ◽  
Helicase Activity ◽  
Single Molecule Fluorescence ◽  
Fluorescence Study

scholarly journals Large domain movements upon UvrD dimerization and helicase activation

2017 ◽  
Vol 114 (46) ◽  
pp. 12178-12183 ◽  
Author(s):  
Binh Nguyen ◽  
Yerdos Ordabayev ◽  
Joshua E. Sokoloski ◽  
Elizabeth Weiland ◽  
Timothy M. Lohman
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Self Assembly ◽  
Total Internal Reflection ◽  
Dna Helicase ◽  
Conformational State ◽  
Helicase Activity ◽  
Multiple Conformations ◽  
Dna Substrate

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

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 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.

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