Conformation of the RNA polymerase II C-terminal domain: circular dichroism of long and short fragments

Abstract

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandemly repeated copies of a heptapeptide with the Y 1S 2P 3T 4S 5P 6S 7 consensus sequence. This repeat contains two overlapping SPXX motifs that can adopt a β-turn conformation. In addition, each CTD repeat contains the PXXP sequence characteristic of the left-handed helix 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 circular dichroism (CD) to characterize the conformation of the CTD in water and in the hydrogen bond-promoting solvent trifluoroethanol (TFE). In water, an eight-repeat fragment is predominantly unordered, but at 32 °C has P II and β-turn contents estimated to be about 15 % and less than 10 %, respectively. In 90 % TFE, the β-turn fraction is estimated to be about 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 α-aminoisobutyric acid leads to a large increase in β-turn. Replacement of Ser2 by Ala does not substantially alter the CTD conformation in water or TFE. Ser5 replacement by Ala increases the P II content in water and affects the conformation in TFE-rich solutions. Phosphorylation of Ser2 and Ser5 has little effect in water, but Ser2 affects the conformation in TFE-rich solution in much the same way as Ser5→Ala substitution. The CD of the full-length murine CTD in water is similar to that of the eight-repeat fragment, indicating little difference in conformation with increasing chain length beyond eight repeats. The roles of P II and β-turn in the interaction of CTD with its target proteins (mediator and RNA-processing components) are discussed. The most likely interactions are between P II and WW or SH3 domains, or with some unknown P II-binding motif.