Cloning, Sequencing, and Transcriptional Analysis of the Gene Coding for the Vegetative Sigma Factor of

The sigA gene of Agrobacterium tumefaciens was cloned and sequenced. Comparison with previously analyzed sigA genes revealed a high degree of similarity in nucleotide and amino acid sequences of regions two, three, and four of vegetative sigma factors. However, the upstream regulatory region shows no sequence homology with the Escherichia coli heat shock (&-32) promoters. It also does not contain the hairpin-loop structure (inverted repeat sequence) that was found in the upstream region of the groE operon in A. tumefaciens. The transcription initiation site of the gene was determined and found to be at the same position during normal growth and under heat shock conditions. Furthermore, no heat shock activation was observed at the transcriptional level. The specificity of transcription initiation in prokaryotes is conferred by the interaction of the RNA polymerase and promoters. The RNA polymerase is a multisubunit enzyme composed of at, IB, I', and sigma subunits. The association of the sigma subunit with the holoenzyme (a2131') confers specificity to the promoters. In Escherichia coli, the vegeta-tive sigma factor is sigma-70 (coded by the rpoD gene), and the heat shock response is mediated by an alternative sigma factor protein: sigma-32 (13, 14). This sigma factor recog-nizes a different promoter sequence and in this way specif-ically transcribes heat shock genes (1, 3, 4). The rpoD gene of E. coli is one of the genes that is transcriptionally activated during heat shock, due to the presence of a promoter recognized by sigma-32 which is located in its regulatory region (8, 17). Recently, it was observed that gram-positive bacteria, such as Bacillus subtilis, and gram-negative bacteria, such as cyanobacteria (Synechococcus spp.) and chlamydiae, con-tain an inverted repeat sequence in the upstream regulatory region of genes coding for the heat shock proteins GroE