COP10 is a ubiquitin-conjugating enzyme variant (UEV), which is thought to act together with COP1, DET1, and the COP9 signalosome (CSN) in Arabidopsis to repress photomorphogenesis. Here, we demonstrate that COP10 interacts with ubiquitin-conjugating enzymes (E2s) in vivo, and can enhance their activity in vitro, an activity distinct from previous characterized UEVs such as MMS2 and UEV1. Furthermore, we show that COP10 forms a complex with UV-damaged DNA-binding protein 1a (DDB1a) and de-etiolated 1 (DET1), and physically interacts with COP1 and the CSN. Purified CDD (COP10, DDB1, DET1) complex also shows enhancement of E2 activity (UEA) similar to that observed with COP10 itself. Our data suggests that COP10, along with COP1 and the CSN, promotes the degradation of positive regulators of photomorphogenesis, such as the transcription factor HY5, via the ubiquitin/26S proteasome system. Thus, the CDD complex may act as a ubiquitylation-promoting factor to regulate photomorphogenesis.

SUMMARY The nucleosome represents a mechanical barrier to transcription that operates as a general regulator of gene expression. We investigate how each nucleosomal component – the histone tails, the specific histone-DNA contacts, and the DNA sequence – contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones. In contrast, point mutations that affect histone-DNA contacts at the dyad abolish the barrier to transcription in the central region by decreasing the local wrapping rate. Moreover, we show that the nucleosome amplifies sequence-dependent transcriptional pausing, an effect mediated through the structure of the nascent RNA. Each of these nucleosomal elements controls transcription elongation by affecting distinctly the density and duration of polymerase pauses, thus providing multiple and alternative mechanisms for control of gene expression by chromatin remodeling and transcription factors.

During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation.DOI: http://dx.doi.org/10.7554/eLife.00971.001

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 ...