The positive control of ilvC expression in E. coli K-12

Abstract

The mechanism of ilvC expression in Escherichia coli was investigated. To carry out this work several different approaches were used. Firstly, sequencing of the ilvY2143 allele which carries a mutation that makes ilvC expression constitutive was completed. The location of the mutation was determined to be at the 5 end of the gene. It is a single base substitution (G to A) at position 87 (counted from the transcription startpoint of ilvY). This results in a change of the codon for one amino acid. Glutamine in wild-type ilvY protein is replaced by lysine in the constitutive one. This substitution in the polypeptide of the upsilon protein (product of the ilvY gene) was found to be solely responsible for making the up silon protein independent of the ilvC gene substrates (a-acetohydroxybutyrate or acetolactate) needed for ilvC induction. Two approaches were used to determine the direction of ilvY trancription. One of these employed a gene fusion technique which involves two DNA fragments of ilvY being fused separately to a promoterless lacZ gene, then monitoring the expression of lacZ. The other approach involved the labeling of the upsilon protein withes -methionine after expression of ilvY in a T7 RNA polymerase dependent promoter system. DNA-binding activity of upsilon protein was investigated. This was carried out in two assays, filter binding and gel retardation assays. These assays were employed to monitor purification of upsilon protein to near homogeneity. Upsilon protein has a subunit size of 35 kd and a native molecular weight of approximately 211 kd, suggesting upsilon exists as a hexamer. Finally, in vitro activities of the upsilon protein were tested using transcriptional and coupled transcription-translation assays. Upsilon protein was shown to cause elevation of ilvC transcription. Two models for the action of the upsilon protein in regulating the transcription of the ilvYC are proposed.