scholarly journals Single-Molecule Studies of RNA Polymerase: One Singular Sensation, Every Little Step It Takes

2011 ◽  
Vol 41 (3) ◽  
pp. 249-262 ◽  
Author(s):  
Matthew H. Larson ◽  
Robert Landick ◽  
Steven M. Block
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Single Molecule Studies

scholarly journals Single Molecule Studies on Hcv RNA Polymerase Activity

2010 ◽  
Vol 98 (3) ◽  
pp. 73a-74a
Author(s):  
Pierre Karam ◽  
Colins Vasquez ◽  
Wayne Mah ◽  
Megan Powdrill ◽  
Matthias Götte ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Polymerase Activity ◽  
Hcv Rna ◽  
Single Molecule Studies ◽  
Rna Polymerase Activity

scholarly journals A Novel Complex: A Quantum Dot Conjugated to an Active T7RNA Polymerase

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Mette Eriksen ◽  
Peter Horvath ◽  
Michael A. Sørensen ◽  
Szabolcs Semsey ◽  
Lene B. Oddershede ◽  
...  
Keyword(s):  
Quantum Dot ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Rna Polymerases ◽  
Fluorescent Signal ◽  
Single Molecule Level ◽  
Single Molecule Studies ◽  
Novel Complex ◽  
Luminescent Nanoparticle

To perform single-molecule studies of the T7RNA polymerase, it is crucial to visualize an individual T7RNA polymerase, for example, through a fluorescent signal. We present a novel complex combining two different molecular functions, an active T7RNA polymerase and a highly luminescent nanoparticle, a quantum dot. The complex has the advantage of both constituents: the complex can traffic along DNA and simultaneously be visualized, both at the ensemble and at the single-molecule level. The labeling was mediated through anin vivobiotinylation of a His-tagged T7RNA polymerase and subsequent binding of a streptavidin-coated quantum dot. Our technique allows for easy purification of the quantum dot labeled T7RNA polymerases from the reactants. Also, the conjugation does not alter the functionality of the polymerase; it retains the ability to bind and transcribe.

scholarly journals Efficient reconstitution of transcription elongation complexes for single-molecule studies of eukaryotic RNA polymerase II

Transcription ◽  
2012 ◽  
Vol 3 (3) ◽  
pp. 146-153 ◽  
Author(s):  
Murali Palangat ◽  
Matthew Larson ◽  
Xiaopeng Hu ◽  
Averell Gnatt ◽  
Steven Block ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcription Elongation ◽  
Single Molecule Studies

scholarly journals Single-Molecule Studies of Human RNA Polymerase Ii Transcription Initiation: Core-Promoter Recognition

2011 ◽  
Vol 100 (3) ◽  
pp. 65a
Author(s):  
Alexandros Pertsinidis ◽  
Sang Ryul Park ◽  
Robert Coleman ◽  
Andrei Revyakin ◽  
Robert Tjian ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Promoter Recognition ◽  
Single Molecule Studies ◽  
Rna Polymerase Ii Transcription

scholarly journals Single molecule studies reveal branched pathways for activator-dependent pre-initiation complex assembly

2021 ◽  
Author(s):  
Inwha Baek ◽  
Larry J. Friedman ◽  
Jeff Gelles ◽  
Stephen Buratowski
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Tata Box ◽  
Initiation Complex ◽  
Kinetic Measurements ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Nucleotide Triphosphates ◽  
Single Molecule Studies

RNA polymerase II (Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.

scholarly journals Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Manchuta Dangkulwanich ◽  
Toyotaka Ishibashi ◽  
Shixin Liu ◽  
Maria L Kireeva ◽  
Lucyna Lubkowska ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcription Elongation ◽  
Energy Landscape ◽  
Ratchet Mechanism ◽  
Elongation Cycle ◽  
Nucleotide Addition ◽  
Single Molecule Studies ◽  
Rate Limiting

During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation.

scholarly journals Single molecule studies of RNA polymerase II transcription in vitro

Transcription ◽  
2014 ◽  
Vol 5 (1) ◽  
pp. e27608 ◽  
Author(s):  
Abigail E Horn ◽  
James A Goodrich ◽  
Jennifer F Kugel
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Single Molecule Studies ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription

scholarly journals Single-Molecule Studies of RNA Polymerase: Motoring Along

2008 ◽  
Vol 77 (1) ◽  
pp. 149-176 ◽  
Author(s):  
Kristina M. Herbert ◽  
William J. Greenleaf ◽  
Steven M. Block
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Single Molecule Studies
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