Biochemical and structural studies have shown that the initiation of RNA polymerase II (pol II) transcription proceeds in the following stages: assembly of pol II with general transcription factors (GTFs) and promoter DNA in a “closed” preinitiation complex (PIC)1,2; unwinding about 15 bp of the promoter DNA to form an “open” complex3,4; scanning downstream to a transcription start site; synthesis of a short transcript, believed to be about 10 nucleotides; and promoter escape. We have assembled a 32-protein, 1.5 megadalton PIC5 derived from Saccharomyces cerevisiae and observed subsequent initiation processes in real time with optical tweezers6. Contrary to expectation, scanning driven by transcription factor IIH (TFIIH)7-12 entailed the rapid opening of an extended bubble, averaging 85 bp, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule opticaltrapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.Pol II | optical tweezers | optical trap T he expression of most genes is carefully regulated at the level of transcription. As a consequence, RNA polymerase II (RNAPII)-the enzyme responsible for mRNA synthesis in eukaryotic organisms-is at the nexus of an exquisite network of regulatory pathways, many of which are controlled by transcription factors. The control pathways associated with RNAPII recruitment to, and initiation at, promoter sites have been studied extensively (1), but it has become increasingly clear that significant regulatory activity also occurs during postinitiation steps and, in particular, at the level of transcript elongation (2).Productive transcript elongation (3-5) is characterized by periods of unidirectional motion by RNAPII along the DNA template, adding one nucleotide at a time to the growing RNA transcript. Elongation-both in vitro in highly purified systems (6, 7) and in vivo (8, 9)-is frequently interrupted by transcriptional pauses, at least some fraction of which are associated with enzyme backtracking, a process by which RNAPII reverses its normal direction of motion and moves upstream on the template (6, 7). Entry into backtracked states appears to confer a high degree of for...

impair electron transport complexes, and lead to neuronal apoptosis. Recently, using a channel reconstitution technique, we demonstrated that monomeric asyn both reversibly blocks and translocates through the voltage-dependent anion channel (VDAC) at nanomolar concentrations. Considering VDAC's major role in regulating metabolite fluxes across the MOM, a functional interaction between a-syn and VDAC could be essential for physiological adaptation of mitochondria and dysfunction in PD. Here we show that the asyn-VDAC interaction is modulated by membrane lipid composition relevant to MOM. We have found that the on-rate of VDAC blockage by a-syn increases up to 10-fold with increase of phosphoethanolamine (PE) content in phosphocholine (PC) membranes. Remarkably, the off-rate was also lipid-dependent, as seen by a 5 mV increase of the turnover potential separating regimes of blockage and translocation with the increase of PE content. We have also found that at physiologically low salt concentrations, the on-rates increase more than 10-fold compared to experiments performed in high salt, while translocation of a-syn through VDAC is impeded. a-Syn differential binding to lipid membranes was also tested using independent methods. These results suggest that the blockage of VDAC by a-syn involves a-syn interaction with the membrane and is governed by both hydrophobic and electrostatic components. Such evidence provides further support for our hypothesis that a-syn needs to bind to the membrane prior to blocking and translocating through the VDAC pore. We propose a new regulatory role of mitochondrial lipids in the (patho-)physiology of monomeric a-syn interaction with mitochondria.

Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the −35 and −10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (−10 to −35) and with the −10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.

During transcriptional elongation, RNA polymerases (RNAP) employ a stepping mechanism to translocate along the DNA template while synthesizing RNA. Optical trapping assays permit the progress of single molecules of RNA polymerase to be monitored in real time, at resolutions down to the level of individual base pairs. Additionally, optical trapping assays permit the application of exquisitely controlled, external forces on RNAP. Responses to such forces can reveal details of the load-dependent kinetics of transcriptional elongation and pausing. Traditionally, the bacterial form of RNAP from E. coli has served as a model for the study of transcriptional elongation using optical traps. However, it is now feasible to perform optical trapping experiments using the eukaryotic polymerase, RNAPII, as well. In this report, we describe the methods to perform optical trapping transcriptional elongation assays with both prokaryotic RNAP and eukaryotic RNAPII. We provide detailed instructions on how to reconstitute transcription elongation complexes, derivatize beads used in the assays, and perform optical trapping measurements.