RNA polymerase II-associated protein (RAP) 74 binds transcription factor (TF) IIB and blocks TFIIB-RAP30 binding

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Transcription Factor IIF (TFIIF) is crucial for RNA polymerase II transcription.
• TFIIF consists of RAP30 and RAP74 subunits, with RAP30 being the smaller subunit.
• Understanding the functional domains of RAP30 is essential for elucidating TFIIF's role in transcription regulation.

Purpose of the Study:

• To map the functional domains of human RNA polymerase II-associated protein 30 (RAP30).
• To investigate the binding interactions between RAP30, RAP74, and TFIIB.
• To elucidate the role of these interactions in the assembly and function of transcription factor IIF (TFIIF).

Main Methods:

• Construction and analysis of deletion mutants of human RAP30.
• In vitro transcription assays to assess functional activity.
• Co-immunoprecipitation or pull-down assays to determine binding interactions between protein subunits.

Main Results:

• Both N-terminal and C-terminal sequences of RAP30 are critical for its function in transcription.
• RAP74 binds to the N-terminal region of RAP30 (amino acids 1-98).
• TFIIB binds to an overlapping region of RAP30 (amino acids 1-176, minimal region 27-152), and RAP74 can block this interaction.

Conclusions:

• The N-terminal domain of RAP30 is essential for RAP74 binding, while both N- and C-terminal regions are important for overall RAP30 function.
• RAP74 directly binds TFIIB via its C-terminal region (amino acids 358-517), and this binding inhibits TFIIB-RAP30 interaction.
• Dissociation of the TFIIF complex may be necessary for physiological TFIIB-RAP30 interactions during transcription.