Requirements of the RNA Polymerase II C-Terminal Domain for Reconstituting Pre-mRNA 3′ Cleavage

Abstract

RNA polymerase II (RNAP II) has previously been shown to be required for the pre-mRNA polyadenylation cleavage reaction in vitro. This activity was found to reside solely in the C-terminal domain (CTD) of the enzyme's largest subunit. Using a deletion analysis of glutathione S-transferase-CTD fusion proteins, we searched among the CTD's 52 imperfectly repetitive heptapeptides for the minimal subset that possesses this property. We found that heptads in the vicinity of 30 to 37 contribute modestly more than other sections, but that no specific subsection of the CTD is necessary or sufficient for cleavage. To investigate further the heptad requirements for cleavage, we constructed a series of all-consensus CTDs having 13, 26, 39, and 52 YSPTSPS repeats. We found that the nonconsensus CTD heptads are together responsible for only 20% of the wild-type cleavage activity. Analysis of the all-consensus CTD series revealed that the remaining 80% of the CTD-dependent cleavage activity directly correlates with CTD length, with significant activity requiring ≈26 or more repeats. These results are consistent with a scaffolding role for the RNAP II CTD in the pre-mRNA cleavage reaction.