Research in the Luse Laboratory

Our laboratory studies transcription from a mechanistic perspective, using the human transcriptional machinery and primarily biochemical approaches. We have recently focused on (i) the processes through which RNA polymerase II (pol II) initiates RNA synthesis and clears the promoter, (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 our studies is a better understanding of the control of gene expression. The accurate duplication of the various stages of transcription in test tube systems provides the necessary tools to uncover regulatory mechanisms.

Transcript initiation and promoter clearance

The pol II preinitiation complex includes a large number of initiation factors in addition to the polymerase itself. The actual roles of these accessory factors in the initiation process and the early stages of transcript elongation are poorly understood. In addition it has not been clear how pol II breaks free from the extensive network of promoter-specific interactions in order to enter into productive transcript elongation. We have addressed these problems in a significant way through several recent discoveries. We showed that promoter escape and the establishment of the nascent elongation complex is closely linked to the propagation of the “transcription bubble” (the melted segment of the DNA template used by the polymerase to access the template strand- see Pal et al., (2005) Mol. Cell 19, 101). The energy released by the reclosure of the upstream half of the initial transcription bubble (bubble collapse) drives the transition from initiation into elongation. Linked to this transition is the displacement of the TFIIB initiation factor (Cabart et al. (2011), ms. under review). We have also mapped the pathway of the nascent RNA as it emerges from pol II and begins to interact with the polymerase surface (Ujvari and Luse (2006) Nat. Struct. Mol. Biol. 13, 49).

Transcript elongation

It has been appreciated for some time the gene expression is not only regulated by controlling the assembly of pol II at promoters but also during transcript elongation. Thus, controlling the action of factors that stimulate elongation is likely to be an important aspect of gene regulation. As part of our studies on the clearance process, we discovered that treatment of pol II initiation complexes with a kinase in nuclear extracts leads to slower rates of subsequent transcript elongation. We found that the kinase in question is casein kinase 2 (CK2) and the modification target is the TFIIF factor. (Note that TFIIF is both required for initiation and stimulatory for elongation.) Remarkably, CK2 phosphorylation prevents TFIIF from facilitating elongation or simply binding to pol II elongation complexes but phospho-TFIIF is fully functional in supporting transcript initiation. These findings, which were recently published (Ujvari et al. (2011) J. Biol. Chem. 286, 23160), suggest an interesting mechanism for maintaining a promoter in an active state while suppressing the ability to make full-length transcripts from the downstream gene.

An unexpected additional discovery emerged from our studies on CK2-modified TFIIF. We have found that while phospho-TFIIF is fully functional in directing the assembly of the pol II initiation complex, phospho-TFIIF does not remain resident in such complexes, contradicting the prevailing view that TFIIF is obligatory for transcript initiation itself (Cabart et al. (2011), ms under review). We also made the unanticipated observation that the stability of TFIIB in the pol II initiation complex depends critically on TFIIF.

Transcription of nucleosomal templates

Our third major project concerns the ability of pol II to elongate nascent RNAs on nucleosomal templates. In the nucleus, template DNA is packed within long arrays of nucleosomes; however, our early studies had shown that even a single nucleosome forms an essentially absolute barrier to transcript elongation by pol II at normal ionic strength. Our recent experiments have employed a more defined in vitro transcription system consisting of a DNA fragment bearing a promoter for pol II and a single, precisely positioned downstream nucleosome. Along with our long-time collaborators, Dr. Vasily Studitsky and colleagues (UMDNJ), we have used this approach to map the location of the major transcriptional barrier within the nucleosome and the pathway through which DNA unfolds from the nucleosome surface to allow the passage of pol II along the template (Bondarenko et al. (2006) Mol. Cell 24, 469). With this model in mind we then showed that alterations in the nucleosome that should specifically assist template unwrapping at the major barrier do considerably facilitate nucleosome traversal by pol II (Ujvari et al., (2008) J. Biol. Chem. 283, 32236; Hsieh et al. (2010) EMBO Reports 11, 705). Most importantly, we have recently demonstrated that combining appropriate combinations of transcript elongation factors with elongation-facilitating nucleosome alterations allows pol II to cross single nucleosomes with the same efficiency, and at nearly the same rate, as pure DNA templates (Luse et al. (2011) J. Biol. Chem. 286, 6040). We hope to build on these recent successes to better understand the role in transcript elongation of additional cellular factors which can influence template unwrapping from the nucleosome surface. Our long-term goal is to demonstrate the rapid and efficient traversal of nucleosome arrays in the test tube, which should now achievable based on our recent work with single nucleosomes.