Mechanical processes are involved in nearly every facet of the cell cycle. Mechanical forces are generated in the cell during processes as diverse as chromosomal segregation, replication, transcription, translation, translocation of proteins across membranes, cell locomotion, and catalyzed protein and nucleic acid folding and unfolding, among others. Because force is a product of all these reactions, biochemists are beginning to directly apply external forces to these processes to alter the extent or even the fate of these reactions hoping to reveal their underlying molecular mechanisms. This review provides the conceptual framework to understand the role of mechanical force in biochemistry.

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...

RNA polymerase is an essential enzyme for cellular gene expression. In an effort to further understand the enzyme's importance in the cell's response to temperature, we have probed the kinetic mechanism of E. coli RNA polymerase (RNAP) by studying the force-velocity behavior of individual RNAP complexes at temperatures between 7°C and 45°C using optical tweezers. Within this temperature range and at saturating nucleotide concentrations, the pause-free transcription velocity of RNAP was independent of force and increased monotonically with temperature with an elongation activation energy of 9.7 ± 0.7 kcal/mole. Interestingly, the pause density at cold temperatures (7 °C to 21 °C) was five times higher than that measured above room temperature. A simple kinetic model revealed a value of 1.29 ± 0.05 kcal/mol for the activation energy of pause entry, suggesting that pause entry is indeed a thermally accessible process. The dwell time distribution of all observable pauses was independent of temperature, directly confirming a prediction of the model recently proposed for Pol II in which pauses are diffusive backtracks along the DNA. Additionally, we find that the force at which the polymerase arrests (the arrest force) presents a maximum at 21 °C, an unexpected result as this is not the optimum temperature for bacterial growth. This observation suggests that arrest could play a regulatory role in vivo, possibly through interactions with specific elongation factors.