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

Abstract

Transcription is the first of many biochemical steps that turn the genetic information found in DNA into the proteins responsible for driving cellular processes. In this review, we highlight certain advantages of single-molecule techniques in the study of prokaryotic and eukaryotic transcription, and the specific aspects in which these techniques complement conventional, ensemble-based biochemistry. We focus on recent literature, highlighting ways in which single-molecule methods have provided fresh insights into mechanism. We also present recent technological advances and outline future directions in the field.

X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

“…For example, active E. coli RNAP can be conveniently reconstituted in vitro from its individual subunits using either wild-type or mutant proteins (14,15), and its mechanism can be easily probed in vitro in the presence of purified template DNA, factors, and transcription factors. A simple and robust E. coli transcription system also makes it an excellent model for single-molecule studies of RNAPs (16).…”

Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies

“…Recently, single-molecule techniques have been used to visualize the movements of RNAP on the template DNA in real time, which has provided kinetic information for almost every step of the transcription cycle 38,39 . In the past two years, exciting progress has been made towards understanding the assembly of initiation complexes, the conformational movements made by RNAP in the course of a transcription cycle and the physical principles underpinning the regulation of transcription initiation.…”

“…Until recently, single-molecule studies of transcription had focused on the movements of RNAP HE relative to the DNA template 38,39 , or unwinding of the template during open-complex formation 46 . In a recent smFRET study, conformational changes in RNAP HE were measured, and together with existing crystal structure data, these measurements provide a dynamic model of RNAP HE structure throughout the transcription cycle 47 .…”

Optical tweezers studies of transcription by eukaryotic RNA polymerases

“…First, these techniques provide the ability to simplify the complex in vivo transcription machinery and to track and manipulate individual RNA polymerase mole cules, one at a time, without the myriad of additional interactions that take place inside a cell (4,5). Second, they enable access to the dynamics of single molecules, and thus allow us to circumvent the necessity to average over thousands of molecules in bulk experiments (4)(5)(6). Third, with single-molecule techniques it is possible to measure highly dynamic and fast movements, given the high spatiotemporal resolution that has become available (7).…”