Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.T ranscription regulation is the first step in the control of gene expression and it is a fundamental and highly coordinated process in all cells. RNA polymerase II (Pol II) is responsible for the synthesis of mRNAs, most snRNAs, and microRNAs in eukaryotic cells. Transcription elongation by Pol II is regulated by many elements such as the state of Pol II phosphorylation in the C-terminal repeat domain (CTD), the presence and stability of nucleosomes, the extent and stability of the nascent RNA structure formed behind Pol II, and several transcription factors. However, many of the molecular details underlying Pol II transcriptional regulation remain unknown.When Pol II synthesizes an RNA transcript, the enzyme translocates along the DNA template by thermally fluctuating between the pre-and the posttranslocated states; the binding of NTP to the posttranslocated state rectifies the forward motion in a mechanism that is consistent with Pol II operating as a Brownian ratchet (1-3). After the binding of an NTP, Pol II rapidly hydrolyzes the NTP, extends the nascent RNA transcript by 1 nt, and releases pyrophosphate (PPi). During transcription elongation, Pol II is also susceptible to entering a paused state, which is a major regulatory element for transcriptional repression (4). In a paused state, Pol II is known to backtrack wherein the 3′ end of the nascent RNA transcript is extruded from its active site. Backtracked Pol II molecules remain catalytically inactive and may become transcriptionally competent only when the enzyme restores the registry between the Pol II's active site and the 3′ end of the transcript either by diffusion forward along the DNA template or by cleaving of the misaligned transcript at the backtracked position of the active site (1, 5).Many transcription factors, including TFIIS and TFIIF, are known to regulate transcription elongation by directly interacting with the polymerase (6). TFIIS rescues backtracked Pol II molecules by stimulating the intrinsic endonucleolytic activity of Pol II (7). An internal scission of the RNA backbone removes 2-nt or longer fragments of the nascent RNA and returns the enzyme to a posttranslocate...

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

SignificanceOptical tweezers enable scientists to follow the dynamics of molecular motors at high resolution. The ability to discern a motor’s discrete steps reveals important insights on its operation. Some motors operate at the scale of angstroms, rendering the observation of their steps extremely challenging. In some cases, such small steps have been observed sporadically; however, the full molecular trajectories of steps and intervals between steps remain elusive due to instrumental noise. Here, we eliminate the main source of noise of most high-resolution dual-trap optical tweezers and developed both a single-molecule assay and a self-learning algorithm to uncover the full trajectories of such a motor: RNA polymerase. Using this method, a whole new set of experiments becomes possible.