scholarly journals One-dimensional sliding assists σ70-dependent promoter binding by Escherichia coli RNA polymerase

2018 ◽  
Author(s):  
Iddo Heller ◽  
Margherita Marchetti ◽  
Abhishek Mazumder ◽  
Anirban Chakraborty ◽  
Agata M. Malinowska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Three Dimensional ◽  
Optical Traps ◽  
Facilitated Diffusion ◽  
One Dimensional ◽  
Single Dna Molecules ◽  
Search Mechanism ◽  
Dna Base

ABSTRACTThe search for a promoter on DNA by RNA polymerase (RNAP) is an obligatory first step in transcription. The role of facilitated diffusion during promoter search has been controversial. Here, we re-assessed facilitated diffusion in promoter search by imaging motions of single molecules of Escherichia coli RNAP σ70 holoenzyme on single DNA molecules suspended between optical traps in a manner that absolutely avoided interactions with surfaces. The assay enabled us to observe unambiguous one-dimensional sliding of RNAP σ70 holoenzyme for thousands of DNA base pairs during promoter search. Analysis of binding kinetics revealed short binding events on nonspecific DNA (0.4 s), intermediate binding events on A/T-rich DNA (1.6 s), and long binding events at or near promoters (>300 s). We estimate a lower bound for the “diffusion facilitation threshold” – the RNAP concentration at which three-dimensional search and one-dimensional sliding contribute equally to promoter binding – of 0.2 μM RNAP. The results suggest facilitated diffusion occurs in promoter search by RNAP, even at the relatively high, 0.2-0.6 μM, concentrations of RNAP in cells.Significance statementThe flow of genetic information from DNA to RNA is of central importance to living systems, and it can only start after an RNA polymerase (RNAP) has found a promoter site. But how does this enzyme find promoter sites on DNA in the first place? In recent years, debate on this topic has favored a promoter search mechanism that is dominated by three-dimensional diffusion of RNAP, rather than by one-dimensional sliding of RNAP on DNA. Here, we designed an improved single-molecule assay that unambiguously revealed extensive one-dimensional sliding of RNAP on DNA. Our results imply that, at the RNAP concentrations in living cells, the promoter-search process is facilitated by one-dimensional sliding on DNA.

scholarly journals The promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion

2012 ◽  
Vol 20 (2) ◽  
pp. 174-181 ◽  
Author(s):  
Feng Wang ◽  
Sy Redding ◽  
Ilya J Finkelstein ◽  
Jason Gorman ◽  
David R Reichman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Three Dimensional ◽  
Dimensional Diffusion ◽  
Search Mechanism

scholarly journals Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Facilitated Diffusion ◽  
One Dimensional ◽  
Bacterial Transcription ◽  
Fluorescence Measurements ◽  
Dimensional Diffusion ◽  
Single Molecule Studies ◽  
Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

scholarly journals Bio-Molecular Applications of Recent Developments in Optical Tweezers

Biomolecules ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 23 ◽  
Author(s):  
Dhawal Choudhary ◽  
Alessandro Mossa ◽  
Milind Jadhav ◽  
Ciro Cecconi
Keyword(s):  
Photonic Crystal ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Continuous Wave ◽  
Imaging Techniques ◽  
Resolving Power ◽  
Optical Traps ◽  
Laser Source ◽  
One Dimensional

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

scholarly journals Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

2014 ◽  
Vol 112 (3) ◽  
pp. 743-748 ◽  
Author(s):  
Yara X. Mejia ◽  
Evgeny Nudler ◽  
Carlos Bustamante
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Elongation Rate ◽  
Nucleoside Triphosphate ◽  
Transcription Rate ◽  
Nucleotide Analogs ◽  
Catalytic Center ◽  
Trigger Loop

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP’s pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.

scholarly journals Partitioning of RNA Polymerase Activity in Live Escherichia coli from Analysis of Single-Molecule Diffusive Trajectories

2013 ◽  
Vol 105 (12) ◽  
pp. 2676-2686 ◽  
Author(s):  
Somenath Bakshi ◽  
Renée M. Dalrymple ◽  
Wenting Li ◽  
Heejun Choi ◽  
James C. Weisshaar
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Polymerase Activity ◽  
Rna Polymerase Activity

scholarly journals Direct Observation of One-Dimensional Diffusion and Transcription by Escherichia coli RNA Polymerase

1999 ◽  
Vol 77 (4) ◽  
pp. 2284-2294 ◽  
Author(s):  
Martin Guthold ◽  
Xingshu Zhu ◽  
Claudio Rivetti ◽  
Guoliang Yang ◽  
Neil H. Thomson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Direct Observation ◽  
One Dimensional ◽  
Dimensional Diffusion
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