Structural studies of RNA polymerase II complexes with human TFIIS and capping enzymes

Transcription is a conserved fundamental cellular process carried out by RNA polymerases (Pol) that results in the synthesis of an RNA transcript. Transcription of protein coding genes by Pol II is the first step in conversion of the genetic information into cellular proteome. The Pol II transcription cycle is divided into three distinct phases of initiation, elongation, and termination. In this dissertation, we provide insights into two essential aspects of elongation: Pol II backtracking and co-transcriptional capping. Backtracking is a result of Pol II trying to overcome barriers like the nucleosome. It results in the nascent RNA being extruded into a pore, causing Pol II arrest. Transcription factor II S (TFIIS), rescues Pol II under such circumstances. TFIIS travels through the Pol II crevice, facilitates RNA cleavage with its domain III in the pore, generating a new 3’ end in Pol II active site. The first manuscript (published) focuses on the details of Pol II passage through the nucleosome with the help of TFIIS, where I resolve the structure of the mammalian Pol II-TFIIS. The structure reveals that the mammalian Pol II has a pre-opened crevice allowing insertion of human TFIIS without structural changes, unlike its yeast counterpart. The TFIIS domain III remains mobile, which might enable regulation of TFIIS entry into Pol II active site. The second manuscript (in-preparation), and the primary focus of this dissertation is the co-transcriptional capping of the 5’ end of the emerging RNA. In humans, all mRNAs carry a 5’ cap composed of a 7-methylguanosine attached via an inverted phosphate to the first transcribed nucleotide (nt) which is methylated at 2’ O. This structure is essential to protect the RNA from exonucleolytic attacks and serves as a marker for ‘self-RNA,’ maintaining an antiviral state in the cell. DRB sensitivity inducing factor (DSIF), an elongation factor facilitates capping. Through this dissertation, I propose the first comprehensive model of mammalian co-transcriptional capping based on six structural intermediates of the process. The structures show that the human capping enzymes use Pol II stalk as a stable anchor to bind and act on the pre-mRNA at the exit channel. These structures enable us to capture the RNA movement between enzyme active sites, and the major structural rearrangements of DSIF that exemplify the importance of its modular nature. Failure to rescue a backtracked Pol II or to incorporate a 5’ cap result in pre-mature transcription termination. In summary, our elucidated structures help unravel the molecular mechanisms governing transcription elongation and provide a robust framework for the fundamental process of co-transcriptional capping.