Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II can be phosphorylated by a p34cdc2/CDC28-containing CTD kinase. Phosphorylated serine (or threonine) is located at positions 2 and 5 in the repetitive heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show here that phosphorylation of the mouse CTD retards its electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels in a way similar to that observed for the II0 form of the largest subunit of RNA polymerase II phosphorylated in vivo. At the maximum level of phosphorylation by CTD kinase in vitro, there are 15-20 phosphates evenly distributed among the 52 heptapeptide repeats that comprise the mouse CTD. Gel filtration chromatography and sucrose gradient ultracentrifugation analyses indicate that phosphorylation induces a dramatic conformational change in the CTD with the phosphorylated form adopting a far more extended structure than the unphosphorylated CTD.     

Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit

The largest subunit of eukaryotic RNA polymerase II contains a carboxyl-terminal domain (CTD) which is comprised of repetitive heptapeptides with a consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We demonstrate here that the mouse CTD expressed in and purified from Escherichia coli can be phosphorylated in vitro by a p34cdc2/CDC28-containing CTD kinase from mouse ascites tumor cells. The product of this reaction, a phosphorylated form of the CTD, contains phosphoserine and phosphothreonine, but not phosphotyrosine. The same phosphoamino acid content is observed in the in vivo phosphorylated CTD from a mouse cell line. Synthetic peptides with naturally occurring non-consensus heptapeptide sequences can also be phosphorylated by CTD kinase in vitro. Phosphoamino acid analysis of these non-consensus heptapeptides together with direct sequencing of a phosphorylated heptapeptide reveals that serines (or threonines) at positions two and five are the sites phosphorylated by mouse CTD kinase. Thus, the -Ser(Thr)-Pro- motif common to p34cdc2/CDC28-containing protein kinases is the recognition site for mouse CTD kinase.     

BRCA1 can modulate RNA polymerase II carboxy-terminal domain phosphorylation levels

A high incidence of breast and ovarian cancers has been linked to mutations in the BRCA1 gene. BRCA1 has been shown to be involved in both positive and negative regulation of gene activity as well as in numerous other processes such as DNA repair and cell cycle regulation. Since modulation of the RNA polymerase II carboxy-terminal domain (CTD) phosphorylation levels could constitute an interface to all these functions, we wanted to directly test the possibility that BRCA1 might regulate the phosphorylation state of the CTD. We have shown that the BRCA1 C-terminal region can negatively modulate phosphorylation levels of the RNA polymerase II CTD by the Cdk-activating kinase (CAK) in vitro. Interestingly, the BRCA1 C-terminal region can directly interact with CAK and inhibit CAK activity by competing with ATP. Finally, we demonstrated that full-length BRCA1 can inhibit CTD phosphorylation when introduced in the BRCA1(-/-) HCC1937 cell line. Our results suggest that BRCA1 could play its ascribed roles, at least in part, by modulating CTD kinase components.     

Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK.

The cyclin-dependent kinase (CDK)-activating kinase, CAK, from mammals and amphibians consists of MO15/CDK7 and cyclin H, a complex which has been identified also as a RNA polymerase II C-terminal domain (CTD) kinase. While the Schizosaccharomyces pombe cdc2 gene product also requires an activating phosphorylation, the enzyme responsible has not been identified. We have isolated an essential S.pombe gene, mop1, whose product is closely related to MO15 and to Saccharomyces cerevisiae Kin28. The functional similarity of Mop1 and MO15 is reflected in the ability of MO15 to rescue a mop1 null allele. This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H-related cyclin Mcs2 of S.pombe. Also, Mop1 and Mcs2 can associate with the heterologous partners human cyclin H and MO15, respectively. Moreover, the rescue of a temperature-sensitive mcs2 strain by expression of mop1+ demonstrates a genetic interaction between mop1 and mcs2. In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. The CAK activity of Mop1-Mcs2 distinguishes it from the related CDK-cyclin pair Kin28-Ccl1 from S.cerevisiae, and supports the notion that Mop1-Mcs2 may represent a homolog of MO15-cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK.     

A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II

The activation of cyclin-dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated. When R2 was overexpressed in a CAK-deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation. Immunoprecipitates of rice proteins with the anti-R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis. Mutational analysis indicated that R2 phosphorylated the threonine residue within the T-loop of CDK2 and Cdc2Os1. R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK- and CTD-kinase activities associated with R2 were identified in a complex of 105 kDa. These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant Arabidopsis.