Biochemical and structural studies of the transcription elongation factor SPT6 and its biding partners

Production of a functional protein requires coordination and regulation of many factors that control different cellular processes. One of these factors, Spt6, is a highly conserved nuclear protein that has roles in several facets of gene expression. Spt6 is best known for its ability to chaperone histones and modulate chromatin structure, but it also functions as a transcription elongation factor and in mRNA processing and export. Spt6 co-localizes with elongating RNA polymerase II (RNAPII) where it reassembles nucleosomes following RNAPII passage in order to repress aberrant transcription initiation. Furthermore, Spt6 directly stimulates RNAPII elongation rates and coordinates co-transcriptional mRNA processing. Despite the wealth of functional data that implicate Spt6 in these processes, little is known about the mechanistic basis for these activities. In order to gain mechanistic understanding of Spt6 activities, the work presented in this thesis focused on biochemical, structural, and functional characterization of Spt6 interactions with other proteins. These studies reveal the true Spt6 binding site on RNAPII, identify Tom1 as a novel Spt6 binding partner, and demonstrate a specific interaction between Spt6 and histones H3-H4 that is competitive with DNA. The elucidation of the authentic Spt6 binding site on RNAPII has allowed us to develop tools to probe the mechanism of Spt6 recruitment to transcribed regions. Identification of Tom1 iv as a phosphorylated Spt6 binding partner provides a physical link to potential Spt6 functions such as mRNA export, cell cycle regulation, and regulation of histone levels. Characterization of the interaction with H3-H4 brings us closer to a mechanistic understanding of the histone chaperone activity of Spt6. Overall, the data presented in this work advance our knowledge of the interactions that regulate Spt6 function and will aid future studies to further dissect the mechanistic basis for Spt6 roles in gene expression.