DNA-binding properties and characterization of human transcription factor TFIIIC2.
Interaction between the B-block region of adenovirus VA1 DNA and the human RNA polymerase III transcription factor (TFIII) C2 was analyzed using a gel DNA-binding assay. The retarded band corresponding to the specific complex between TFIIIC2 and the regulatory B-block region was identified by DNase I footprint analysis, competition experiments, and gel shift assays using mutated and truncated virus-associated (VA) 1 DNA probes. The equilibrium constants for the binding reaction with the complete VA1 gene were determined. TFIIIC2 was found to bind to non-specific DNA sequences with a relatively low affinity (equilibrium constant Kn = 6 x 10(4) M-1), and to the B-block sequence with a high affinity (specific constant Ks = 2 x 10(11) M-1). Assuming one site per molecule, the total concentration of binding sites [C0] in the TFIIIC2-containing fractions ranged between 0.6 and 1.6 x 10(-10) M. This corresponded to 1500 TFIIIC2 molecules extracted per 293 cell. Sedimentation analysis of TFIIIC2 on a sucrose gradient showed both VA1 DNA binding activity and in vitro transcription activity cosedimenting with an apparent coefficient of 17-18 S, consistent with a molecular weight of 400-500 kDa. UV cross-linking of a 5-bromo 2'-deoxyuridine-containing, 32P-labeled VA1 probe with the TFIIIC2 fraction, followed by DNase I digestion and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed a single 32P-labeled band migrating as a 250-kDa polypeptide. Compared with the sedimentation data, this result suggests that native TFIIIC2 may be a dimer of the approximately 250-kDa polypeptide.
Boulanger
P A
PA
Molecular Biology Institute, University of California, Los Angeles 90024.
Yoshinaga
S K
SK
Berk
A J
AJ
D000260:Adenoviruses, Human / Q000235:genetics*; D001665:Binding Sites; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005814:Genes, Viral; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D011485:Protein Binding; D012320:RNA Polymerase III / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC
D004279:DNA, Viral; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III