Escherichia coli RNA polymerase translocates along the DNA discontinuously during the elongation phase of transcription, spending proportionally more time at some template positions, known as pause and arrest sites, than at others. Current models of elongation suggest that the enzyme backtracks at these locations, but the dynamics are unresolved. Here, we study the role of lateral displacement in pausing and arrest by applying force to individually transcribing molecules. We find that an assisting mechanical force does not alter the translocation rate of the enzyme, but does reduce the efficiency of both pausing and arrest. Moreover, arrested molecules cannot be rescued by force, suggesting that arrest occurs by a bipartite mechanism: the enzyme backtracks along the DNA followed by a conformational change of the ternary complex (RNA polymerase, DNA and transcript), which cannot be reversed mechanically.E scherichia coli RNA polymerase (RNAP) is a highly processive enzyme responsible for the transcription of DNA into RNA. The ternary elongation complex of DNA, RNAP and RNA is extremely stable, with RNAP capable of reaching speeds of 35 nucleotides per second as it translocates along the DNA (1). Despite this rapid translocation during elongation, RNAP is sensitive to the sequence it transcribes, displaying temporary (pauses) and permanent (arrests) halts to transcription, which are believed to play a role in the regulation of gene expression (2).Transcriptional pauses and arrests can occur by various mechanisms but share common features. These features include the continued stability of the ternary elongation complex (3) and the displacement of the RNA 3Ј end from the enzyme active site (4, 5). Kinetic evidence suggests that both pauses and arrests are kinetically off the main elongation pathway, and that pauses are intermediate between elongation and arrest states (4, 6-8):Much of the information about pauses and arrests has come from biochemical footprinting and crosslinking studies on complexes walked to particular template positions. These studies have suggested that core RNA polymerase is, variously, a flexible enzyme, capable of undergoing inchworming motion (9-11); a rigid enzyme, capable of monotonic translocation (12); and a ''sliding clamp'' enzyme, capable of frequent backwards and forwards oscillations (13-15). Among these proposed models, the importance given to an RNA:DNA hybrid and to different protein-nucleic acid interactions to explain the stability of the complex varies (16). Within the sliding clamp model, proteinnucleic acid contacts confer stability to the ternary complex (through a protein clamp enclosing downstream DNA), whereas the RNA:DNA hybrid (of 8-12 bp) is responsible for accurately positioning the enzyme active site with the 3Ј OH of nascent RNA (13,15,17). The recently obtained crystal structure of a bacterial RNA polymerase shows evidence of a downstream DNAbinding site, and suggests that an RNA:DNA hybrid of 8-9 bp is easily accommodated within the enzyme, which is consistent wi...

In Xenopus laevis, transcription of the gamma-fibrinogen subunit gene is activated by glucocorticoids. Hormone induction is regulated by three glucocorticoid response element (GRE) half-sites and an additional DNA sequence which binds a novel hepatocyte nuclear protein, Xenopus glucocorticoid receptor accessory factor (XGRAF). The XGRAF binding site (GAGTTAA) is located directly upstream of the most distal half-GRE. The proximity of the binding sites for XGRAF and the glucocorticoid receptor (GR) led to the hypothesis that these two sites form a glucocorticoid response unit (GRU). By transfecting DNA into primary hepatocytes, we showed that this GRU confers hormone responsiveness in the absence of other half-GREs. The XGRAF binding site enhances function of the half-GRE without itself responding to glucocorticoids. The GRU retains efficacy in other locations relative to the gamma-fibrinogen gene promoter, further increases transcription when present in multiple copies, and activates a heterologous promoter. Despite the contiguity of the XGRAF binding site and half-GRE, the two sites can be occupied simultaneously in vitro. The binding characteristics correlate with function since mutations that disrupt concurrent XGRAF and GR binding also impair transcription. This novel GRU represents a new regulatory mechanism that may be applicable to other glucocorticoid responsive genes that lack a full GRE.

The adrenal steroid hormones, glucocorticoids, control many physiological responses to trauma, including elevated synthesis of fibrinogen, a major blood-clotting protein. Glucocorticoid regulation of the gamma-fibrinogen subunit gene in Xenopus laevis is mediated by a binding site for Xenopus glucocorticoid receptor accessory factor (XGRAF) and a contiguous glucocorticoid response element (GRE) half-site. Here, we characterize the protein:DNA complex formed by a cooperative interaction between XGRAF, GR, and the DNA. We demonstrate that the complex contains XGRAF by competition in a gel shift assay. The presence of GR is established by two criteria: 1) size dependence of the XGRAF:GR:DNA complex on the size of the GR component and 2) interference with complex formation by GR antibody. Cooperative binding of XGRAF and GR to the DNA was quantitated, showing that GR favors binding to XGRAF:DNA compared with free DNA by a factor of 30. The cooperative interaction between XGRAF and GR can occur on nicked DNA but is disrupted when 1 bp is inserted between the XGRAF binding site and half-GRE. Significantly, this loss of physical association in vitro correlates with loss of XGRAF amplification of GR activity in transiently transfected primary Xenopus hepatocytes. The simplest explanation for cooperativity between XGRAF and GR is formation of a DNA-bound heterodimer of these two proteins. This mechanism represents a new mode of transcriptional regulation in which GR and a nonreceptor protein form a heterodimer, with both partners contacting their specific DNA sites simultaneously.