scholarly journals Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies

2014 ◽  
Vol 42 (8) ◽  
pp. 5177-5190 ◽  
Author(s):  
Amit Sharma ◽  
Robert N. Leach ◽  
Christopher Gell ◽  
Nan Zhang ◽  
Patricia C. Burrows ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Active Site ◽  
Sigma Factor ◽  
Dna Delivery ◽  
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

Single-molecule studies of DNA and RNA four-way junctions

2004 ◽  
Vol 32 (1) ◽  
pp. 41-45 ◽  
Author(s):  
S.A. McKinney ◽  
E. Tan ◽  
T.J. Wilson ◽  
M.K. Nahas ◽  
A.-C. Déclais ◽  
...  
Keyword(s):  
Single Molecule ◽  
Active Site ◽  
Dynamic Structure ◽  
Holliday Junctions ◽  
Site Formation ◽  
Structural Element ◽  
Fluorescence Studies ◽  
Definitive Evidence ◽  
Dna And Rna ◽  
Single Molecule Studies

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.

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 Relevance of single cell and single molecule studies at different biological and physical length scales

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Quantum Mechanics ◽  
Single Cell ◽  
Single Molecule ◽  
Active Site ◽  
Covalent Bond ◽  
The Other ◽  
Quantum Mechanical ◽  
Microfluidic Channels ◽  
Other Hand ◽  
Single Molecule Studies

Scale transcends multiple levels of biological and physical organization, and is the critical factor that determines success of any scientific investigation. Specifically, the scale at which a question is posed plays a crucial role in influencing the type of experiments and apparatuses needed. Single cell and single molecule experiments came to the fore of experiment science due to its capability at addressing a fundamental problem in biology and physical science: does the same behavior in cells and molecules transcend different length and population scales? Thus far, single cell experiments could be achieved with trapping of single cell using optical tweezer traps and microfluidic channels. The same, however, is not true for single molecule studies, which remains in the realm of theoretical and simulation studies. Specifically, single molecule experiment remains at the hundreds to thousands of molecules level, where possible skew in the population of molecules sampled could provide a false depiction of molecular reality of a larger population. But, what do scientists learn from single cell and single molecule studies? Is it the uncovering of mysteries of the probabilistic behavior at the single entity level, guided by perhaps quantum mechanics? The answer is no for single cell studies, given that cellular decision making require the input of tens to hundreds of molecular sensors and effectors within a cell. Hence, single cell behavior is not random, but directed at a nutrient or concentration gradient or signaling source. On the other hand, enzymatic catalysis of a single molecule substrate with the active site involves a quantum mechanical crosstalk. Thus, reaction between the substrate molecule and the active site proceeds if suitable energy levels (i.e., quantum mechanical states) are found for both parties. Given that distribution of quantum mechanical states is probabilistic, stochasticity rules single molecule interaction such as a covalent bond formation reaction between reactant A and B. Thus, single cell and single molecule studies do hold relevance in biological and physical sciences research if the correct experiment tool is used for a pertinent question at an appropriate length and population scale. For example, while tremendous amount of basic understanding could be derived from single cell experiments, single cell perspective is not relevant to questions examining the interactions between two large subpopulations of cells. Single molecule experiments, on the other hand, remains in the theoretical and simulation realm for highlighting the effect of quantum mechanics in guiding the behavior of molecules at the nanoscale.

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 Relevance of single cell and single molecule studies at different biological and physical length scales

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Quantum Mechanics ◽  
Single Cell ◽  
Single Molecule ◽  
Active Site ◽  
Covalent Bond ◽  
The Other ◽  
Quantum Mechanical ◽  
Microfluidic Channels ◽  
Other Hand ◽  
Single Molecule Studies

Scale transcends multiple levels of biological and physical organization, and is the critical factor that determines success of any scientific investigation. Specifically, the scale at which a question is posed plays a crucial role in influencing the type of experiments and apparatuses needed. Single cell and single molecule experiments came to the fore of experiment science due to its capability at addressing a fundamental problem in biology and physical science: does the same behavior in cells and molecules transcend different length and population scales? Thus far, single cell experiments could be achieved with trapping of single cell using optical tweezer traps and microfluidic channels. The same, however, is not true for single molecule studies, which remains in the realm of theoretical and simulation studies. Specifically, single molecule experiment remains at the hundreds to thousands of molecules level, where possible skew in the population of molecules sampled could provide a false depiction of molecular reality of a larger population. But, what do scientists learn from single cell and single molecule studies? Is it the uncovering of mysteries of the probabilistic behavior at the single entity level, guided by perhaps quantum mechanics? The answer is no for single cell studies, given that cellular decision making require the input of tens to hundreds of molecular sensors and effectors within a cell. Hence, single cell behavior is not random, but directed at a nutrient or concentration gradient or signaling source. On the other hand, enzymatic catalysis of a single molecule substrate with the active site involves a quantum mechanical crosstalk. Thus, reaction between the substrate molecule and the active site proceeds if suitable energy levels (i.e., quantum mechanical states) are found for both parties. Given that distribution of quantum mechanical states is probabilistic, stochasticity rules single molecule interaction such as a covalent bond formation reaction between reactant A and B. Thus, single cell and single molecule studies do hold relevance in biological and physical sciences research if the correct experiment tool is used for a pertinent question at an appropriate length and population scale. For example, while tremendous amount of basic understanding could be derived from single cell experiments, single cell perspective is not relevant to questions examining the interactions between two large subpopulations of cells. Single molecule experiments, on the other hand, remains in the theoretical and simulation realm for highlighting the effect of quantum mechanics in guiding the behavior of molecules at the nanoscale.

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 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 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
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