Mutational analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein.
RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5.
Le
Thi Thu Thuy
TT
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan.
Zhang
Shijun
S
Hayashi
Naoyuki
N
Yasukawa
Mami
M
Delgermaa
Luvsanjav
L
Murakami
Seishi
S
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D019473:Transcription Factors, TFII; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D012319:RNA Polymerase II; C056604:transcription factor TFIIF