Structural and biochemical studies of the transcription termination machinery

Abstract

RNA Polymerase II (PolII) dependent transcription of mRNAs is central to gene expression throughout eukaryotes. Transcription is a highly regulated process, with a defined initiation, elongation, and termination phase, all of which are controlled by multiple trans-acting protein factors and cis-acting elements on the template DNA and transcribed RNA. Extensive work has shown that the phosphorylation state of the C- terminal domain (CTD) of PolII plays a central role in the recruitment of trans-acting factors during all phases of transcription. Aberrant phosphorylation can result in a lethal phenotype in yeasts, therefore implying that correct control of phosphorylation by kinases and phosphatases specific for the CTD is critical for life. In addition to the polymerase, conserved cis-acting sequence elements near the 3'-end on pre-mRNAs help to define and recruit various RNA processing machines to the transcription elongation complex in order to properly process and package the pre-mRNA for export to the cytoplasm for translation. In this thesis, I first review current knowledge regarding PolII CTD phosphorylation and its effects on the transcription elongation complex. Additionally, in this first chapter, I will also provide an overview of the 3'-end mRNA processing/transcription termination machinery. In the second part of my thesis, I will describe my doctoral work on the structural and biochemical characterization of Rtr1, a unique PolII CTD phosphatase that represents a novel new member of this class of enzymes. In my studies, I show that Rtr1 is a bona fide phosphatase of unique sequence and structure that is allosterically regulated by its own C-terminus. Additionally, I show that Rtr1 is a dual specificity phosphatase, with activities against both serine and tyrosine residues on the CTD. In chapter 3 of this thesis, I describe my work on the in vitro reconstitution of the Cleavage Stimulation Factor (CstF) responsible for the recognition of sequences downstream of the polyadenylation site on pre-mRNAs that help to define the 3'-end processing reaction that occur at the end of genes. Using highly purified proteins, I show for the first time that CstF is a dimer of trimers, with two copies of each subunit in the entire assembly. In addition, I show that CstF, as a complex, can bind to G/U rich RNAs with nanomolar affinities, in stark contrast with previous studies showing that singly purified proteins from the complex binding with much weaker affinities.