Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP).
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.
National Key Laboratory of Medical Neurobiology, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China. weiwe@umdnj.com
Wenxiang
W
Wei
Jun Xia
JX
Gu
Cui Qing
CQ
Zhu
Feng Yan
FY
Sun
Dorjbal
D
Dorjsuren
Yong
Y
Lin
Seishi
S
Murakami
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics*; D005057:Eukaryotic Cells / Q000378:metabolism*; D005809:Genes, Regulator / Q000235:genetics*; D016153:Genes, Suppressor / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D017434:Protein Structure, Tertiary / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins; D019473:Transcription Factors, TFII / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D019473:Transcription Factors, TFII; C117184:URI1 protein, human; D012319:RNA Polymerase II; C056604:transcription factor TFIIF