CHARACTERIZATION OF THE METR BINDING-SITES FOR THE GLYA GENE OF ESCHERICHIA-COLI

Sequence analysis of the glyA control region of Escherichia coli ident ified two regions with homology to the consensus binding sequence for MetR, a lysR family regulatory protein, Gel shift assays and DNase I p rotection assays verified that both sites bind MetR. Homocysteine, a c oregulator for MetR, increased MetR binding to the glyA control region , The MetR binding sites were cloned into the pBend2 vector. Although the DNA did not show any significant intrinsic bend, MetR binding resu lted in a bending angle of about 33 degrees, MetR-induced bending was independent of homocysteine. To verify that the MetR binding sites pla y a functional role in glyA expression, site-directed mutagenesis was used to alter the two binding sites in a lambda glyA-lacZ gene fusion phage. Changing the binding sites toward the consensus MetR binding se quence caused an increase in glyA-lacZ expression, Changing either bin ding site away from the consensus sequence caused a decrease in expres sion, suggesting that both sites are required for normal glyA regulati on.