Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

Abstract

RNA polymerase pausing represents an important mechanism of transcriptional regulation. In this study, we use a single-molecule transcription assay to investigate the effect of template base-pair composition on pausing by RNA polymerase II and the evolutionarily distinct mitochondrial polymerase Rpo41. For both enzymes, pauses are shorter and less frequent on GC-rich templates. Significantly, incubation with RNase abolishes the template dependence of pausing. A kinetic model, wherein the secondary structure of the nascent RNA poses an energetic barrier to pausing by impeding backtracking along the template, quantitatively predicts the pause densities and durations observed. The energy barriers extracted from the data correlate well with RNA folding energies obtained from cotranscriptional folding simulations. These results reveal that RNA secondary structures provide a cis-acting mechanism by which sequence modulates transcriptional elongation.enzyme kinetics | Pol II | transcriptional pausing | yeast T ranscription represents the first point of control of gene expression. Its regulation determines the RNA levels in the cell and, ultimately, such varied processes as cell-cycle coordination, metabolism, growth, and death. Regulation of RNA throughput occurs at all stages of the transcription process, i.e., initiation, elongation, and termination (1). Regulation at initiation involves complex machinery that assembles at the promoter and endows the cell with the capacity to make a binary decision in response to internal or external signals. Similarly, transcriptional termination requires a binary decision at specific locations in the sequence. In contrast, regulation of the elongation phase is spatially distributed throughout the transcribed gene and involves the modulation of the dynamics of RNA synthesis by cis-and trans-acting factors.One of the most prominent aspects of the dynamics of RNA polymerases is their tendency to pause. Pausing is an intrinsic property of most RNA polymerases, and its regulation constitutes one of the central mechanisms of control of gene expression. Sequence-specific pausing allows for the recruitment of trans-acting elements that are implicated in promoter escape (2), alternative splicing (3), factor-dependent termination (4), proofreading (5), and further transcriptional regulation (6-9). Pauses also play a role in factor-independent termination, which is mediated by secondary structures of the nascent RNA near the termination site (10). In contrast to the punctate nature of pausing during initiation and termination, pauses during elongation can, in general, occur anywhere along the transcribed gene.The finding that pause durations of RNAP II follow a t −3∕2 distribution (to first order) implies that pausing occurs through a diffusive mechanism (5). This observation, along with additional evidence of backward movement of RNA polymerases on their template (11,12), has led to the backtracking model of transcriptional pausing. In this model, pauses are initiated by retrograde movement ...

Optical tweezers studies of transcription by eukaryotic RNA polymerases

“…It was proposed that the secondary structure of the transcribed RNA presents an energetic barrier for polymerase backtracking, thus preventing the polymerase to go into a deep backtrack and reducing the overall backtracking time (67). Theoretical predictions were confirmed experimentally in an optical tweezers study that showed that the GC content of the DNA template and hence, the nascent RNA structure, affects the pausing dynamics of RNA polymerases, with polymerases pausing less on the GC-rich templates that are known to form more secondary structures (34). The energy barrier to backtracking due to the secondary structure of the RNA is also considered in a recent theoretical study, where the entropy produced during backtracking is discussed (68).…”

“…Depending on the experimental system, i.e. assisting or opposing force mode and the concentration of nucleotides used, the measured velocity ranged between 15 and 25 nt/s (9,11,21,22,(33)(34)(35).…”

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation

“…The rate of RNAP II backtracking in the absence of RNA 3′ end retraction is about one nucleotide per 0.3 s, about 20-fold slower than forward polymerization. 96 Assuming a similar rate for RNAP III, for the ~20% of tRNA genes that have a predicted structure 4-5 nts upstream of the T tract, the required backtracking would represent a relatively slow step. For the dozens of genes with hairpins farther upstream, 6-12 nts, 13 the required backtracking would be more time consuming.…”

Single molecule studies of RNA polymerase II transcription in vitro

“…27 It was hypothesized that secondary structure in the nascent RNA would prevent RNAP II backtracking by presenting an energy barrier to backward movement of the polymerase. RNAP II pause duration and density was compared on GC-rich vs. AT-rich templates; transcripts made from the GC-rich templates would form stronger hairpins.…”