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.
ABSTRACTrRNA synthesis decreases significantly during the differentiation ofrat L6 myoblasts to myotubes. Nuclear run-on assays demonstrated that the decrease was attributable to decreased rates of rRNA gene transcription. Immunoblot analysis indicated a marked reduction in amounts of the RNA polymerase I transcription factors UBF1 and UBF2 (upstream binding factors 1 and 2, respectively). The levels of these factors dropped in parallel with the down-shift in rRNA gene transcription. The amount of UBF does not fail due to a general decrease in cellular protein, as myosin heavy-chain protein accumulates markedly during this same time. RNA blots of total RNA isolated from myoblasts and differentiating myotubes showed a decrease in the mRNA for UBF, at the same time the mRNA for myogenin was accumulating. The downshift in UBF mRNA levels preceded the decrease in the protein levels for UBF. There have been reports that the acute response of the rRNA gene transcription system to physiological signals in many systems involves an RNA polymerase I-associated factor. However, our results imply that the regulation of rRNA gene DNA transcription in response to physiological processes, such as differentiation, may involve multiple regulatory pathways.
The DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of cyanobacteria contains a unique core component, -y, which is absent from the RNA polymerases of other eubacteria (G. J. Schneider, N. E. Tumer, C. Richaud, G. Borbely, and R. Haselkorn, J. Biol. Chem. 262:14633-14639, 1987). We present the complete nucleotide sequence of rpoCl, the gene encoding the -y subunit, from the heterocystous cyanobacterium Nostoc commune UTEX 584. The derived amino acid sequence of -y (621 residues) corresponds with the amino-terminal portion of the ,' polypeptide of Escherichia coli RNA polymerase. A second gene in N. commune UTEX 584, rpoC2, encodes a protein which shows correspondence with the carboxy-terminal portion of the E. coli f1' subunit. The rpoBCIC2 genes of N. commune UTEX 584 are present in single copies and are arranged in the order rpoBCIC2, and the coding regions are separated by short AT-rich spacer regions which have the potential to form very stable secondary structures. Our data indicate the occurrence of divergent evolution of structure in the eubacterial DNA-dependent RNA polymerase.The transcription of genes is directed through the activity of DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). In eubacteria, a single form of the core RNA polymerase, together with ancilliary sigma factors, is responsible for the synthesis of virtually all cellular RNAs (5). The RNA polymerase of Escherichia coli consists of at least four different subunits, 3, P', a, and ur, and is present in two main enzyme forms, core (W'%a2) and holoenzyme (core plus cr; 4). The two genes encoding the P (rpoB) and 3' (rpoC) subunits of this RNA polymerase are adjacent to one another and are cotranscribed from the major promoter PL10 (5). The basic (P3'a2) design has been found in the RNA polymerases purified from representatives of gram-positive and gramnegative eubacteria (17, 38). Recently, however, an additional core component, y, has been described for the RNA polymerase (Py,Y'a2ofC of the cyanobacterium Anabaena sp. strain PCC 7120 (32). The -y subunit is serologically unrelated to the other subunits of the cyanobacterial RNA polymerase, but anti-y serum cross-reacts with both E. coli pI' subunit protein and subunit A of the RNA polymerase from Sulfolobus acidocaldarius, an archaebacterium (31). The -y subunit has since been detected in the RNA polymerases of 15 out of 15 taxonomically diverse cyanobacteria, including two Nostoc species (31).Three different nuclear RNA polymerases are found in eucaryotes, each one responsible for the transcription of a different class of genes (17). Comparison of the amino acid sequences of the largest subunit, A, of RNA polymerases II and III from Saccharomyces cerevisiae and the P' subunit of the E. coli RNA polymerase revealed six regions (I to VI) of marked conservation (1).The RNA polymerases of archaebacteria appear to be more closely related to those of eucaryotes (6,38 whether archaebacteria, like...
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