Purification and characterization of proximal sequence element-binding protein 1, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter.
The promoter structure of the known small nuclear RNA (snRNA) genes contains two major effectors of transcriptional activity, a proximal sequence element (PSE) and a distal sequence element (DSE). In previous work, methidiumpropyl-EDTA-Fe(II) footprinting was used to demonstrate the existence in human placental extracts of a protein producing footprints within the PSE and the DSE of the human U1 snRNA gene. This protein (PSE1) has now been purified to homogeneity from both human placental extract and K562 cell nuclear extract. PSE1 consists of two subunits, an alpha subunit with an apparent molecular mass of 83 kDa, and a beta subunit with an apparent molecular mass of 73 kDa in K562 nuclear extracts and 63 kDa in placental extracts. Footprinting and UV cross-linking assays indicate that purified PSE1 binds to the PSE and DSE of the U1 gene. Monoclonal antibodies were prepared which specifically recognize the individual subunits of PSE1. PSE1 is immunologically similar to and shares amino acid sequence with a protein (TREF) which binds the human transferrin receptor (HTFR) promoter. An in vitro transcription system was established for a template consisting of a minimal HTFR promoter placed upstream of the human U1 snRNA-coding region and shown by immunodepletion/addback experiments to specifically require PSE1. Transcription from the adenovirus 2 major late promoter was unaffected in these experiments. This result supports a functional role of PSE1 as a transcriptional activating protein, but its role in transcription of snRNA genes remains to be established. PSE1 also has an immunological relationship to and shares amino acid sequence with the p70 and p86 subunits of the human Ku autoantigen. Ku, PSE1, and TREF may thus be identical proteins or members of a family of heterodimeric proteins consisting of related subunits. Our results support earlier proposals that Ku may be a transcriptional activator.
Knuth
M W
MW
Department of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Gunderson
S I
SI
Thompson
N E
NE
Strasheim
L A
LA
Burgess
R R
RR
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D000954:Antigens, Surface / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D006801:Humans; D000072200:Ku Autoantigen; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011506:Proteins; D012342:RNA, Small Nuclear / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D034961:Antigens, Nuclear; D000954:Antigens, Surface; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D011506:Proteins; D012342:RNA, Small Nuclear; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen