Journal Article

Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP)

WEI, Wenxiang, ZHU, Cui Qing, SUN, Feng Yan, MURAKAMI, Seishi, DORJSUREN, Dorjbal, GU, Jun Xia, LIN, Yong

2003

Overview

RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase II as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74 of TFIIF subunits. In the immunoprecipitation assay in COS1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-amino-acid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.

• Publisher: Nature Publishing Group,National Key Laboratory of Medical Neurobiology, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China%Department of Hematology, the Affliated Xiehe Hospital, Tongji Medical School, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, China%Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan
• Subject: Animals / Binding Sites - genetics / Carrier Proteins - genetics / COS Cells / DNA-directed RNA polymerase / Elongation / Eukaryotic Cells - metabolism / Genes, Regulator - genetics / Genes, Suppressor - physiology / Immunoprecipitation / Intracellular Signaling Peptides and Proteins / Mutants / Protein Structure, Tertiary - genetics / RNA Polymerase II - genetics / RNA, Messenger - biosynthesis / RNA, Messenger - genetics / Transcription factors / Transcription Factors, TFII - genetics / Transcription initiation / Transcription initiation factor TFIIB / Transcription, Genetic - genetics / Transcriptional Activation - genetics