Analysis of the phosphorylation, DNA-binding and dimerization properties of the RNA polymerase I transcription factors UBF1 and UBF2.
The phosphorylation, DNA-binding and dimerization properties of both forms of the RNA polymerase I transcription factor UBF were studied and compared. Tryptic peptide maps of in vivo 32P-labeled UBF contained four phospho-peptides. Two of these peptides are predicted to derive from the serine-rich, carboxyl-terminal of UBF. This region contains nine consensus phosphorylation sites for casein kinase II, and is one of the regions phosphorylated in vitro by casein kinase II. Analysis of the DNA-binding properties of recombinant forms of UBF1 and UBF2 by Southwestern blots revealed: (1) a role for the NH2-terminal 102 amino acid domain of UBF1/UBF2 in DNA-binding; (2) the importance of the bases from -106 to -101 of the rat ribosomal DNA promoter for the binding of UBF; and (3) functional differences between UBF1 and UBF2. Glutaraldehyde cross-linking and overlay assays using recombinant forms of UBF1 and UBF2 demonstrated that the molecules can form both homodimers and heterodimers. These assays also demonstrated that the NH2-terminal 102 amino acids of UBF plays a significant role in dimerization and that other domains contribute to dimerization. The dimerization properties of recombinant forms of UBF1 and UBF2 were different, suggesting that the HMG box 2 of UBF1, which is partially deleted in UBF2, also contributes to UBF dimerization.
O'Mahony
D J
DJ
Geisinger Clinic, Sigfried and Janet Weis Center for Research, Danville, PA 17822-2618.
Smith
S D
SD
Xie
W
W
Rothblum
L I
LI
D000818:Animals; D001483:Base Sequence; D047388:Casein Kinases; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D035463:Pol1 Transcription Initiation Complex Proteins; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D012318:RNA Polymerase I / Q000378:metabolism*; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D014157:Transcription Factors; C063844:transcription factor UBF; D004247:DNA; D011494:Protein Kinases; D047388:Casein Kinases; D012318:RNA Polymerase I; D005976:Glutaral