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

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.