Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules

Abstract

Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogenous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a ϳ2650-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, ‫؍‬ 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.Different individual molecules within a purified enzyme or ribozyme preparation can display greatly differing rate constants for catalytic turnover (1-3). Some of this variability may be due to the presence of enzyme molecules with differing covalent structures; these may arise from the presence of two or more related genes, from alternative splicing of a single pre-mRNA, or from post-translational protein modifications (4). However, some heterogeneity may also arise from the adoption by enzyme molecules with identical covalent structures of different conformational states that persist through multiple catalytic turnovers (5, 6). It has been proposed that such conformational heterogeneity may be a heretofore unappreciated feature of macromolecular catalysts in general (2).Bacterial RNA polymerases (RNAPs), 1 and their structurally and mechanistically similar homologs, eukaryotic RNAP II enzymes, are large, multisubunit proteins that synthesize all cellular mRNAs (7,8). These enzymes are processive; a given RNA molecule is synthesized in its entirety by the same RNAP molecule. In bacterial RNAPs, most RNA synthesis takes place in a stable transcription elongation complex (TEC) containing the core RNAP subunits, the template DNA, and the nascent RNA transcript. The TEC structure has been extensively characterized (9 -12). A variety of proteins and DNA sequence elements in both prokaryotes and eukaryotes regulate gene expression by altering the extent to which active TECs terminate transcription upstream of or within genes (13-15). The efficiency of termi...

Stochastic kinetics of ribosomes: Single motor properties and collective behavior

“…There are some similarities between template-dictated polymerizations driven by ribosome and RNAP. The run time distribution of RNAP has been measured and found to be Gaussian [59]. This is consistent with the Gaussian run time distribution for ribosomes predicted in this paper which follows from very general arguments based on the central limit theorem.…”

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…Single-molecule experiments have shown that RNAP advances by one base at a time along DNA, with translocation tightly coupled to RNA synthesis, and that it likely operates by a Brownian ratchet-type mechanism (7-9). Single-molecule experiments have identified and characterized long-lived heterogeneities in RNAP conformations (24), elongation rates (12,13,101), and pause propensities (10). Other single-molecule studies have found that transcriptional initiation involves scrunching of the DNA template until contacts with the promoter are released (5,6), and supplied evidence for an elongation-incompetent intermediate state preceding transcriptional termination (14).…”

“…One early single-molecule study, which was conducted at comparatively low spatial and temporal resolution, reported that wild-type RNAP can spontaneously switch velocity states (100). However, subsequent studies have failed to confirm any such switching (12,13,101), casting doubt on the finding. To date, only one other observation of velocity-state switching has been reported, in this case for an RNAP mutant bearing a point mutation in the β-subunit (but not for the wild type) (13).…”

“…This suggests that the source of heterogeneity may be structural in origin, perhaps due to minor defects in RNAP caused by translation errors, post-translational modifications of RNAP, or different conformers in RNAP folding (101). Supporting the structural origin of heterogeneity, the variance in FRET distances between the RNA 5′ end and a labeled base located on the template DNA in a TEC was significantly larger than the corresponding variance in a control sample with the identical dyes placed on a DNA molecule.…”

Altering the interaction between σ 70 and RNA polymerase generates complexes with distinct transcription-elongation properties

“…Second, RNA polymerase can pause for a longer time and then backtrack (33), which is a behavior that may be part of the proofreading mechanism of the enzyme (34). Last, the diversity in transcription rates (Ն5-fold) of single molecules far exceeds that expected for a single transcribing species (35). Because each molecule maintains its rate for the duration of the measurements (Ϸ5 min), this variation is not due to rapidly interconverting conformers.…”