NMR solution structure of the Set2 SRI domain and preparation of RNA polymerase II complexes with the elongation factor Spt4-Spt5

Vojnic, Erika (2006): NMR solution structure of the Set2 SRI domain and preparation of RNA polymerase II complexes with the elongation factor Spt4-Spt5. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

RNA polymerase II (RNAP II) transiently associates with many different proteins and multiprotein complexes during the mRNA transcription cycle, which includes three phases, initiation, elongation, and termination. This thesis describes structural studies of two factors that facilitate transcription through chromatin. The heterodimeric Saccharomyces cerevisiae elongation factor Spt4-Spt5 (human DSIF) has been identified by biochemical and genetic approaches to help RNAP II transcribe through chromatin. It is assumed that Spt4-Spt5 pauses RNAP II to open a time window for capping enzyme recruitment and addition of a cap to the 5'-end of the nascent RNA. The preparation of milligram quantities of soluble Spt4-Spt5 variants that are suited for structural studies has been achieved. Several strategies to resolve the structure of the RNAP II–Spt4-Spt5 complex were unsuccessful, possibly indicating an intrinsic flexibility of the complex. In addition, there is now evidence for direct links between chromatin modification and transcription elongation. A major player in this process is the histone lysine methyltransferase Set2 which has a modular structure. The catalytic activity of Set2 is mediated by the SET [Su(var)3-9, Enhancer of Zeste, Trithorax] domain. During mRNA elongation, the SRI (Set2 Rpb1-interacting) domain of Set2 binds to the phosphorylated CTD (carboxyl-terminal domain) of RNAP II. The NMR solution structure of yeast Set2 SRI domain has been determined. The structure reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in sigma factors that mediate transcription initiation in bacteria (domain sigma2 in sigma70). NMR titration experiments show that the SRI domain binds a Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues. Amino acid residues that show strong chemical shift perturbations upon CTD binding cluster in two regions on the SRI surface. The results will enable a detailed analysis of the specific CTD interactions underlying the coupling of transcription and chromatin modification by Set2.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: RNA polymerase II, Spt4-Spt5, Set2 SRI domain, NMR,
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 7. Dezember 2006
• 1. Bericht­erstatter:in: Cramer, Patrick