Analysis of upstream activation of thevnfHpromoter ofAzotobacter vinelandii

Abstract

BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, where as addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.Key words: Azotobacter vinelandii, vnfA, vnfH, promoter-lacZ fusion, DNA bending.