Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III.
The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription. We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF gamma and delta subunits. Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies. Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta. Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro. In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations. Along with our previous demonstration of both physical interactions between PTF gamma/PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF.
Bai
L
L
Laboratory of Biochemistry and Molecular Biology, Rockcfeller University, New York, New York 10021, USA.
Wang
Z
Z
Yoon
J B
JB
Roeder
R G
RG
D000595:Amino Acid Sequence; D001402:B-Lymphocytes; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D012318:RNA Polymerase I / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; C101746:SNAPC3 protein, human; D014157:Transcription Factors; D012319:RNA Polymerase II; D012318:RNA Polymerase I