Abstract
Transitions between the different stages of the RNAPII transcription cycle involve the recruitment and exchange of factors, including mRNA capping enzymes, elongation factors, splicing factors, 3'-end-processing complexes, and termination factors. These transitions are coordinated by the dynamic phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNAPII (Rpb1). The CTD is composed of reiterated heptapeptide repeats (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) that undergo phosphorylation and dephosphorylation as RNAPII transitions through the transcription cycle. An essential phosphatase in this process is Ssu72, which exhibits catalytic specificity for Ser(P)(5) and Ser(P)(7). Ssu72 is unique in that it is specific for Ser(P)(5) in one orientation of the CTD and for Ser(P)(7) when bound in the opposite orientation. Moreover, Ssu72 interacts with components of the initiation machinery and affects start site selection yet is an integral component of the CPF 3'-end-processing complex. Here we provide a comprehensive view of the effects of Ssu72 with respect to its Ser(P)(5) phosphatase activity. We demonstrate that Ssu72 dephosphorylates Ser(P)(5) at the initiation-elongation transition. Furthermore, Ssu72 indirectly affects the levels of Ser(P)(2) during the elongation stage of transcription but does so independent of its catalytic activity.
Highlights
Ssu72 is a RNAPII C-terminal domain (CTD) phosphatase; its function in the transcription cycle has not been established
This study is focused on the function of the Ssu72 RNAPII CTD phosphatase in the RNAPII transcription cycle
We have assayed the presence of RNAPII and the phosphorylation status of its CTD using three sets of isogenic ssu72 mutants: (i) one depleted of Ssu72, which correlates with depletion of the hypophosphorylated form of RNAPII and a marked increase in the Ser(P)5 form of RNAPII in whole cell extracts; one expressing stable but catalytically inactive Ssu72; and one expressing Ssu72 with diminished catalytic activity
Summary
Ssu is a RNAPII CTD phosphatase; its function in the transcription cycle has not been established. Transitions between the different stages of the RNAPII transcription cycle involve the recruitment and exchange of factors, including mRNA capping enzymes, elongation factors, splicing factors, 3-end-processing complexes, and termination factors. These transitions are coordinated by the dynamic phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNAPII (Rpb). The levels of Ser(P) were reported to accumulate only at the 3Ј-ends of genes in ssu mutants, suggesting that the phosphatase activity of Ssu acts on Ser(P) during the elongation-termination stage of the transcription cycle (21). We demonstrate an unanticipated function for Ssu in regulation of Ser phosphorylation status, a function that is independent of Ssu catalytic activity
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