Balanced branching in transcription termination

Harrington, Kevin J.; Laughlin, R. B.; Liang, Shoudan

doi:10.1073/pnas.240431598

Cited by 14 publications

(13 citation statements)

References 58 publications

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“…Distribution of Transcription Rates-Data from both single molecule and bulk experiments have been interpreted as evidence for the existence of two (or more) subpopulations of TECs (23,52) that move at rates differing by a factor of ϳ2-fold (27) or more (22). The comparatively large number of single-molecule elongation rates measured here provides a clear picture of the population distribution of rates (Fig.…”

Section: Improved Methods For Measuring the Distribution Of Singlemolementioning

confidence: 66%

“…Allosteric regulation of E. coli RNAP by specific transcript sequences is well established (19,61,62), but the transcription template used here encodes no known regulatory sequences. On the other hand, the origin of kinetic differences between molecules might be purely conformational; it has been speculated that RNAP can adopt a large number of long-lived conformational substates with differing catalytic properties (23). However, direct experimental evidence for purely conformational heterogeneity is lacking.…”

Section: Possible Structural Origins Of Tec Kinetic Heterogeneitymentioning

confidence: 99%

“…performed on a molecular ensemble) kinetics experiments. These studies (17-19, 21, 22) have been interpreted as supporting complex mechanisms in which individual enzyme molecules can switch between two (or more) distinct, parallel reaction pathways by binding an allosteric regulator (17)(18)(19) or through conformational changes much slower than enzymatic turnover (23). However, uncertainty in the interpretation of macroscopic data can arise if the enzyme preparation contains molecular subpopulations with permanent differences in structure and kinetics.…”

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confidence: 99%

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

Nørrelykke

Engh

Landick

et al. 2004

Journal of Biological Chemistry

102

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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...

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Section: Improved Methods For Measuring the Distribution Of Singlemolementioning

confidence: 66%

Section: Possible Structural Origins Of Tec Kinetic Heterogeneitymentioning

confidence: 99%

mentioning

confidence: 99%

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

Nørrelykke

Engh

Landick

et al. 2004

Journal of Biological Chemistry

102

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“…RNAP does not make two coincident DNA contacts, a second conceptually simple explanation would be that the enzyme undergoes a long-lived conformation change during transcription through an upstream sequence that facilitates the termination response at a downstream sequence. This is not an original idea, [44][45][46] but currently lacks a mechanistic explanation and has been challenged previously by a model in which the elongating RNAP rapidly exchanges between a number of different conformations at every template position. 36 In this equilibrium model, the RNAP structure is essentially re-set after each incorporation event.…”

Section: Discussionmentioning

confidence: 99%

Archaeal RNA Polymerase is Sensitive to Intrinsic Termination Directed by Transcribed and Remote Sequences

Santangelo

Reeve

2006

Journal of Molecular Biology

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“…This work was motivated by the possibility that persistent conformational heterogeneity in RNAP might explain the known variation in termination efficiency (Harrington et al, 2001). Pasman and von Hippel measured the termination efficiency (TE) of complexes that had been subjected to various selection steps in order to enrich putative subpopulations.…”

Section: Heterogeneous Behaviormentioning

confidence: 99%

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

2011

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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.

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Balanced branching in transcription termination

Cited by 14 publications

References 58 publications

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

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

Archaeal RNA Polymerase is Sensitive to Intrinsic Termination Directed by Transcribed and Remote Sequences

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

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