Cloning of the RNA polymerase alpha subunit gene from Thermus thermophilusHB8 and characterization of the protein

The region containing the RNA polymerase alpha subunit (RNAP alpha) gene (r poA) and the ribosomal protein genes of a thermophilic eubacterial strain, Thermus thermophilus (Tt) HB8, was cloned from a genomic DNA library by Sou thern hybridization, The gene order in this region is rp136-rps13-rps11-rps 4-rpoA-rpl17, which is identical to that in some other eubacteria. The rpoA gene encodes a 315 amino acid residue protein with a molecular weight of 3 5,013, the amino acid sequence showing 42% identity to that of Escherichia coli (Ec). From the results of comparison of the amino acid sequence and th e predicted secondary structure of the C-terminal domain of Tt RNAP alpha ( Tt alpha CTD) with those of Ec, the overall folding is expected to be simil ar. However, amino acid residues Asn268 and Cys269 in Ec alpha CTD, which a re essential for its interaction with DNA or regulatory proteins, were repl aced by His and Ser, respectively, in Tt alpha CTD. By means of a T7-based expression system in Ec cells, Tt RNAP alpha was overexpressed and purified . The high thermostability of Tt RNAP alpha was demonstrated by the CD spec tra.