Diversity in the rates of transcript elongation by single RNA polymerase molecules.
Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.
Tolić-Nørrelykke
Simon F
SF
Niels Bohr Institute, Blegdamsvej 17, DK-2100 Copenhagen, Denmark.
Engh
Anita M
AM
Landick
Robert
R
Gelles
Jeff
J
D001360:Avidin / Q000737:chemistry; D019921:Biotinylation; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D015233:Models, Statistical; D016011:Normal Distribution; D012319:RNA Polymerase II / Q000737:chemistry; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D013997:Time Factors; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry
D035602:Transcriptional Elongation Factors; D001360:Avidin; D004247:DNA; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases