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.

A review of the various aspects of transcription that have been addressed using methods of single-molecule detection and manipulation is studied. Whereas single-subunit viral polymerases such as T7 and SP6 RNAP can start transcription at a promoter region without additional cofactors, multi subunit bacterial and eukaryotic RNA polymerase (RNAPs) require transcription factors that aid the enzyme to recognize and bind to the promoter. Through the combination of single molecule manipulation and single-molecule fluorescence methods in the same experiment, it should be possible to follow, the internal dynamics of the polymerase or the binding of a regulatory factor and simultaneously monitor the mechanical variables of position, force, and torque. The result of these efforts will be a multidimensional picture of transcription that will provide crucial information about the relative timing of various molecular events and therefore reveal their causal connection

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.

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.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.