Molecular cloning and characterization of the cDNA encoding the largest subunit of mouse RNA polymerase I

We describe the cloning and analysis of mRPA1, the cDNA encoding the largest subunit (RPA194) of murine RNA polymerase I. The coding region comprises an open reading frame of 5151?bp that encodes a polypeptide of 1717 amino acids with a calculated molecular mass of 194?kDa. Alignment of the deduced protein sequence reveals homology to the β′ subunit of Escherichia coli RNA polymerase in the conserved regions a-h present in all large subunits of RNA polymerases. However, the overall sequence homology among the conserved regions of RPA1 from different species is significantly lower than that observed in the corresponding β′-like subunits of class II and III RNA polymerase. We have raised two types of antibodies which are directed against the conserved regions c and f of RPA194. Both antibodies are monospecific for RPA194 and do not cross-react with subunits of RNA polymerase II or III. Moreover, these antibodies immunoprecipitate RNA polymerase I both from murine and human cell extracts and, therefore, represent an invaluable tool for the identification of RNA polymerase I-associated proteins.