Backtracking by single RNA polymerase molecules observed at near-base-pair resolution
Reads0
Chats0
TLDR
Backtracking events occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.Abstract:
Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo1. Its low error rate may be achieved by means of a ‘proofreading’ mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3′ end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events (∼5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.read more
Citations
More filters
Journal ArticleDOI
Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy
Keir C. Neuman,Attila Nagy +1 more
TL;DR: These techniques are described and illustrated with examples highlighting current capabilities and limitations of single-molecule force spectroscopy.
Journal ArticleDOI
Real-Time Kinetics of Gene Activity in Individual Bacteria
TL;DR: This work directly demonstrates transcriptional bursting in Escherichia coli, similar to that indirectly inferred for eukaryotes, and extends protein-based approaches by counting the integer-valued number of transcript with single-molecule resolution.
Journal ArticleDOI
Recent Advances in Optical Tweezers
TL;DR: Although technical in nature, these developments have important implications for the expanded use of optical tweezers in biochemical research and thus should be of general interest.
Journal ArticleDOI
Direct observation of base-pair stepping by RNA polymerase
Elio A. Abbondanzieri,William J. Greenleaf,Joshua W. Shaevitz,Robert Landick,Steven M. Block +4 more
TL;DR: It is concluded that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA, and the force–velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations is determined.
Journal ArticleDOI
Nascent transcript sequencing visualizes transcription at nucleotide resolution
TL;DR: An approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3′ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution is presented, revealing pervasive polymerase pausing and backtracking throughout the body of transcripts.
References
More filters
Journal ArticleDOI
Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization
Ahmet Yildiz,Joseph N. Forkey,Sean A. McKinney,Taekjip Ha,Taekjip Ha,Yale E. Goldman,Paul R. Selvin,Paul R. Selvin +7 more
TL;DR: The results strongly support a hand-over-hand model of motility, not an inchworm model, which moves processively on actin.
Journal ArticleDOI
Biological applications of optical forces
Karel Svoboda,Steven M. Block +1 more
TL;DR: Theories and Applications of Picotensiometry, Foundations of Trup Stiffness Measurements, and more.
Journal ArticleDOI
Stretching DNA with optical tweezers
TL;DR: Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control, suggesting that the intrinsic persistence length remains close to 40 nm.
Journal ArticleDOI
DNA Replication Fidelity
TL;DR: Current understanding of replication fidelity is reviewed, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity.
Journal ArticleDOI
DNA replication fidelity.
TL;DR: This article focuses on the mechanisms that are responsible for high fidelity of replications with the special emphasis on structural studies showing important conformational changes after substrate binding, and stresses the importance of hydrogen bonding, base pair geometry, polymerase DNA interactions and the role of accessory proteins in replication fidelity.