DISSECTION OF THE TRANSCRIPTION MACHINERY FOR HOUSEKEEPING GENES OF BRADYRHIZOBIUM-JAPONICUM

By using a PCR approach, the Bradyrhizobium japonicum sigA gene, which encodes the primary RNA polymerase sigma factor, sigma(80), was clone d and its nucleotide sequence was established. The deduced protein is highly homologous to the SigA protein of Rhizobium meliloti (72% amino acid sequence identity) but less so to RpoD of Escherichia coli (51% identity), Well conserved is the C-terminal end of the protein, which is probably involved in promoter recognition and binding of the RNA po lymerase core enzyme, A remarkable feature of the primary sequence is an alanine- and proline-rich segment of 24 amino acids between conserv ed regions 1 and 2, which might function as an interdomain linker. We purified the B. Japonicum RNA polymerase holoenzyme, One of the subuni ts had an apparent molecular mass of 90 kDa and corresponded to the si gA gene product, as judged by N-terminal amino acid sequencing. The pu rified RNA polymerase was used in an in vitro transcription system to determine the transcription start sites of the rm and groESL(4) operon s. They were identical to those previously identified in vivo. The rm promoter was cloned upstream of a rho-independent terminator, yielding a transcript of about 240 bases, This served as a suitable template t o analyze promoter activity, Then mutant derivatives of the rm promote r were constructed and tested in in vitro transcription experiments, S everal base pairs essential for promoter activity were thus identified , The results suggest that the well-characterized -35/-10 promoter cla ss is predominantly used in B. japonicum for the expression of ''house keeping'' genes.