Research

Our laboratory studies RNA polymerase II. We are currently focusing on (i) the process by which the RNA polymerase begins transcription and clears the promoter; that is, the transition from initiation to the chain elongation phase of transcription, (ii) the molecular mechanisms involved in transcript elongation, and (iii) the effects of chromatin structure on transcript elongation. All of these aspects of transcription are important checkpoints in the regulation of gene expression in the cell. The ultimate goal of these studies is to achieve a more comprehensive picture of the transcription process through the accurate duplication of cellular events in test tube systems.

Our promoter clearance project has its roots in studies of the properties of newly initiated RNA polymerase II complexes. That work led to the finding that such newly-initiated polymerases have a unique property: they are generally prone to fall into a transcriptionally inactive state called transcriptional arrest (see 3-7, below). In comparison, transcription complexes with more than ~50 bases of nascent RNA are not arrest prone except at very rare, specific sequences (see 9, below). We believe that the reluctance of newly-initiated pol II complexes to commit to processive transcript elongation is related to the regulated escape of RNA polymerases from the promoters of many cellular genes such as hsp70 or c-myc.

In the course of our recent investigation of the earliest stages of transcription, we found that newly-initiated RNA polymerase II has the capacity to reposition the RNA-DNA hybrid to a more upstream location on templates (that is, slip backwards) in initially-transcribed regions that have short, directly repeated sequence elements (see 6, below). This transcript slippage reaction has proven to be a very useful tool for investigating the earliest stages of transcription. Determining the distance downstream of transcription start at which the transcript slippage reaction declines and ultimately disappears has provided insight into the physical changes in the RNA polymerase that accompany the commitment to transcript elongation (see 8, below). We have recently discovered an important underlying relationship between the propagation of the “transcription bubble” (the melted segment of the DNA template used by the polymerase to access the template strand), and promoter clearance itself (see 11, below). These insights were achieved through the study of promoter clearance as a function of promoter architecture. This unique approach has allowed us to: (i), establish that clearance itself coincides with an important structural transition, namely the abrupt reclosure of the upstream half of the initial transcription bubble (bubble collapse); (ii), show that bubble collapse, and thus clearance, requires a transcript of at least 7 nt, which ties directly into recent speculations about the likely interaction of TFIIB and RNA beginning at about +6 or +7, and (iii), demonstrate that early transcription complexes with identical transcripts can have very different stabilities depending on how far how they have progressed towards promoter clearance. This last point, which has never been reported before, is in strong contrast to published observations on elongation complexes. It emphasizes the unique nature of the preclearance complex.

Another important aspect of our research has been mapping the path of the nascent RNA as it emerges from within the RNA polymerase. To perform these studies we have crosslinked radiolabeled transcripts to proteins within the transcription complex using UV light. We initially found that the 5' end of the RNA crosslinks very effectively to a component of the splicing machinery once the RNA chain is >24 bases long (see 10, below). More recently, we have identified a crosslink between the nascent RNA and Rpb7, one of the small subunits of RNA polymerase II. This observation has allowed us to more accurately map the exit path of the nascent RNA (see 12, below). Interactions of the Rpb7 subunit with regulatory proteins could represent one mechanism for influencing both RNA processing and transcript elongation.

We have recently published a study on a novel template sequence that poses a barrier to transcript elongation by RNA polymerase II even after the polymerase has reached the mature elongation stage (see 9, below). This site has proven to be very interesting because the sequences at the point of arrest display no recognizable homology to other well-characterized arrest sites. The novel site also contains a distinct upstream sequence element which appears to be present in other arrest sites, although its existence was not noted in earlier studies. We expect that our work in this area will provide new insights into the mechanism of transcriptional processivity and arrest.

Our other major project concerns the ability of RNA polymerase II to elongate nascent RNAs on nucleosomal templates. We had shown (see 1, below) that nucleosomal arrays assembled from purified histones form an essentially absolute barrier to transcript elongation by RNA polymerase II at normal ionic strength. This barrier could be overcome at higher salt concentrations. We had also collaborated with another group to demonstrate that the FACT complex can partially relieve the nucleosomal transcription barrier at normal ionic strength (see 2, below). We have now greatly extended these results with a more defined in vitro transcription system consisting of a DNA fragment bearing a promoter for RNA polymerase II and a single nucleosome downstream. This arrangement allows a precise assessment of the nature of the nucleosomal barrier. We are performing these experiments as a collaborative study with the laboratory of Dr. Vasily Studitsky, at the Robert Wood Johnson School of Medicine, University of Medicine and Dentistry of New Jersey. Very recently, we have applied this approach to nucleosomes assembled over three different positioning sequences, all of which are known to have particularly high affinity for the histone core of the nucleosome. We found that all of these nucleosomes can provide an exceptionally strong blockade of pol II transcript elongation which is resistant to FACT, higher salt concentrations and all known RNA polymerase II elongation factors (see 13, below). However, the additional and surprising observation is that these exceptional blockades are observed in only one transcriptional orientation. Each of these nucleosomes provides only an average transcriptional barrier when crossed in the opposite direction. Thus, the nucleosomal barrier can be polar and all nucleosomes do not provide equivalent transcriptional barriers. In the near future we will extend these studies to explore the role of histone modifications on the ability of RNA polymerase II to elongate transcripts on nucleosomal templates.