Mutational analysis of the transcriptional regulator GcvA: amino acids important for activation, repression, and DNA binding

Abstract

The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon. Random and site-directed PCR mutagenesis was used to create nucleotide changes in gcvA to identify residues of the protein involved in activation, repression, and DNA binding. Single amino acid substitutions at L30 and F31 cause a defect in activation of a gcvT-lacZ fusion but have no effect on repression or DNA binding. Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA. A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a gcvT-lacZ fusion. The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.

FIG. 1

The serine-glycine pathway in E. coli. The genes and the gene products are as follows: serA, 3-phosphoglycerate dehydrogenase; serC, 3-phosphoserine aminotransferase; serB, 3-phosphoserine phosphatase; glyA, serine hydroxymethyltransferase; and gcv, glycine cleavage enzyme system. THF, tetrahydrofolate.

FIG. 2

Putative H-T-H DNA binding domain of GcvA. The amino acids that are part of the H-T-H are denoted as letters inside the boxes and the amino acid changes made are shown below the boxes. The recognition helix likely interacts with target DNA and the stabilizing helix secures the recognition helix in the proper conformation once bound to the target DNA.

FIG. 3

GMS assay for the binding of wt and mutant GcvA proteins to the gcv control region. A 759-bp DNA fragment containing the gcv control region was ³²P-labeled and incubated with twofold serial dilutions of purified mutant or wt GcvA protein. The unbound ³²P-labeled gcv DNA fragment is indicated. Complexes A and B indicate GcvA bound to sites 2 and 3, and sites 1, 2, and 3 of the gcv control region, respectively (see Fig. 4). Band X is an anomalous band seen only with the GcvA V32A protein. Lane 1, no protein; lanes 2 to 4, 10.0, 5.0, and 2.5 nM wt GcvA, respectively; lanes 5 to 7, 10.0, 5.0, and 2.5 nM L30A GcvA, respectively; lanes 8 to 10, 10.0, 5.0, and 2.5 nM F31A GcvA, respectively; lanes 11 to 13, 10.0, 5.0, and 2.5 nM F31L GcvA, respectively; lanes 14 to 16, 10.0, 5.0, and 2.5 nM V32A GcvA, respectively; lanes 17 to 19, 10.0, 5.0, and 2.5 nM S38P GcvA, respectively.

FIG. 4

Protection from DNase I digestion of the gcv control region by wt and V32A GcvA proteins. A 759-bp ³²P-labeled DNA fragment containing the gcv control region was incubated with twofold serial dilutions of wt and V32A gcvA proteins (Materials and Methods) and digested with DNase I. The partial digestion products were run on a denaturing 5% polyacrylamide gel adjacent to the Maxam-Gilbert A + G sequencing reactions. Lane 1, no protein; lanes 2 to 5, V32A GcvA at 50, 100, 200, and 400 nM, respectively; lane 6, A + G sequencing reaction; lane 7, no protein; lanes 8 to 11, wt GcvA at 25, 50, 100, and 200 nM, respectively.