Conformation of the RNA polymerase IIC-terminal domain: Circular dichroismof long and short fragments

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II con sists of tandemly repeated copies of a heptapeptide with the (YSPTSPS7)-S-1 -P-2-T-3-S-4-P-5-S-6 consensus sequence. This repeat contains two overlappi ng SPXX motifs that can adopt a beta-turn conformation. In addition, each C TD repeat contains the PXXP sequence characteristic of the left-handed heli x of polyproline II (P-II) found in SH3 domain ligands and the PXY sequence that is the target for WW domains. We have studied CTD fragments using cir cular dichroism (CD) to characterize the conformation of the CTD in water a nd in the hydrogen bond-promoting solvent trifluoroethanol (TFE). In water, an eight-repeat fragment is predominantly unordered, but at 32 degrees C h as P-II and beta-turn contents estimated to be about 15 % and less than 10 %, respectively. In 90 % TFE, the beta-turn fraction is estimated to be abo ut 75 %, the remainder being unordered and P-II conformations. The Tyr side -chains are ordered to a significant extent in 90 % TFE. Replacement of the fully conserved Pro residues by alpha-amino-isobutyric acid leads to a lar ge increase in beta-turn. Replacement of Ser2 by Ala does not substantially alter the CTD conformation in water or TFE. Ser5 replacement by Ala increa ses the P-II content in water and affects the conformation in TFE-rich solu tions. Phosphorylation of Ser2 and Ser5 has little effect in water, but Ser 2 affects the conformation in TFE-rich solution in much the same way as Ser 5 --> Ala substitution. The CD of the full-length murine CTD in water is si milar to that of the fight-repeat fragment, indicating little difference in conformation with increasing chain length beyond eight repeats. The roles of P-II and beta-turn in the interaction of CTD with its target proteins (m ediator and RNA-processing components) are discussed. The most likely inter actions are between P-II and WW or SH3 domains, or with some unknown P-II-b inding motif. (C) 2000 Academic Press.