Probing the mechanics of the complete DNA transcription cycle in real-time using optical tweezers

TY - JOUR

T1 - Probing the mechanics of the complete DNA transcription cycle in real-time using optical tweezers

AU - Baumann, Christoph G

AU - Cross, Stephen J

PY - 2011

Y1 - 2011

N2 - RNA polymerase (RNAP) is a DNA-dependent motor protein that links ribonucleotide polymerization to force generation and DNA translocation through its active site, i.e., mechanical work. Single-molecule studies using optical tweezers have allowed researchers to probe the load-dependent ribonucleotide incorporation rate and processivity of both single-subunit viral and multisubunit prokaryotic and eukaryotic RNAPs engaged in transcription elongation. A single-molecule method is described here, which allows the complete transcription cycle (i.e., promoter binding, initiation, elongation and termination) to be followed in real-time using dual-trap optical tweezers and a unique "three-bead" geometry. This single-molecule transcription assay can be used to probe the mechanics of both stationary and moving RNAP-DNA complexes engaged in different stages of transcription.

AB - RNA polymerase (RNAP) is a DNA-dependent motor protein that links ribonucleotide polymerization to force generation and DNA translocation through its active site, i.e., mechanical work. Single-molecule studies using optical tweezers have allowed researchers to probe the load-dependent ribonucleotide incorporation rate and processivity of both single-subunit viral and multisubunit prokaryotic and eukaryotic RNAPs engaged in transcription elongation. A single-molecule method is described here, which allows the complete transcription cycle (i.e., promoter binding, initiation, elongation and termination) to be followed in real-time using dual-trap optical tweezers and a unique "three-bead" geometry. This single-molecule transcription assay can be used to probe the mechanics of both stationary and moving RNAP-DNA complexes engaged in different stages of transcription.

UR - http://www.scopus.com/inward/record.url?scp=80054724203&partnerID=8YFLogxK

U2 - 10.1007/978-1-61779-261-8_12

DO - 10.1007/978-1-61779-261-8_12

M3 - Article

C2 - 21809207

SN - 1940-6029

VL - 778

SP - 175

EP - 191

JO - Methods in molecular biology (Clifton, N.J.)

JF - Methods in molecular biology (Clifton, N.J.)

ER -