| Description |
Eukaryotic transcription and mRNA processing depend upon the coordinated interactions of many proteins, including Spn1 and Spt6, which are conserved across eukaryotes, are essential for viability, and associate with each other in some of their biologically important contexts. Spt6 functions at several important regulatory steps in transcription, including nucleosome reassembly, transcription elongation, and mRNA processing and export. As a histone chaperone, Spt6 is important for reassembly of nucleosomes in the wake of elongating RNA polymerase II, a process that is required to regulate transcription initiation and prevent inappropriate transcription from repressed promoters as well as cryptic intragenic transcription start sites. In conjunction with Spt6, Spn1 coordinates the recruitment of mRNA processing and export factors, such as Yra1 and the exosome, thereby enabling biogenesis of mature and export competent mRNA molecules. The functional relevance of Spn1 and Spt6 in chromatin organization and mRNA maturation is well established, although mechanistic details of how these processes are performed are poorly understood. In order to enhance our understanding of the molecular details of Spn1 and Spt6 functions, this thesis has focused on structural, biochemical and functional studies of Spn1 and Spt6 from Saccharomyces cerevisiae. iv The work presented in this dissertation establishes the structures of the entire ordered region of the Spt6 protein, the ordered core of the Spn1 protein, and the Spn1 core in complex with the binding determinant of Spt6. Additionally, we demonstrate the capacity of Spt6 to interact with factors that very likely influence Spt6 function, including histones and nucleosomes. The structures and the functional data described in this dissertation have enhanced our understanding of how Spt6 binds and chaperones histones, and describes a novel role for Spn1 in regulating the histone chaperone activity of Spt6. The Spt6 and Spn1 structures and the biochemical assays developed in this work will aid in future functional and mechanistic studies that will aim to develop a complete molecular and mechanistic model for each Spt6 and Spn1 function. |
