Functional interaction of human Ssu72 with RNA polymerase II complexes

Area of Science:

• Molecular Biology
• Gene Expression Regulation

Background:

• RNA polymerase II C-terminal domain (CTD) phosphorylation is crucial for transcription.
• Kinases and phosphatases dynamically regulate CTD phosphorylation throughout the transcription cycle.
• The precise role of phosphatases in early transcription stages, including preinitiation complex (PIC) formation and initiation, remains unclear.

Purpose of the Study:

• To identify the specific phosphatase responsible for early transcription cycle activity.
• To elucidate the timing and conditions of Ssu72 phosphatase function during transcription initiation and early elongation.
• To investigate the regulation of Ssu72 activity in relation to RNA length and transcription complex composition.

Main Methods:

• Biochemical assays to identify phosphatase activity associated with early elongation complexes.
• Co-immunoprecipitation to confirm physical association of Ssu72 with transcription complexes.
• In vitro transcription assays to assess Ssu72 function under varying conditions (e.g., salt concentration, RNA length).

Main Results:

• Ssu72 was identified as the magnesium-independent phosphatase active on early transcription complexes.
• Ssu72 physically associates with preinitiation complexes and very early elongation complexes, stable to high salt concentrations.
• Ssu72 activity is restricted on complexes with RNA longer than 28 nt, a cutoff that is lost when PICs are washed before initiation.

Conclusions:

• Ssu72 functions during the transition from transcription initiation to early elongation.
• Ssu72 activity is tightly regulated, potentially by factors present in the PIC, suggesting a role prior to or during initiation.
• These findings provide new insights into the dynamic regulation of RNA polymerase II CTD phosphorylation by phosphatases in early transcription.