REGULATION OF THE EXPRESSION OF THE ESCHERICHIA COLI K-12 ILVC GENE

ALAN JAY BIEL, Purdue University

Abstract

In vivo and in vitro studies of the ilvC gene of Escherichia coli were undertaken to elucidate the mechanism of ilvC regulation. Both the in vivo and in vitro studies presented here were facilitated by the isolation of an ilvC-lac fusion strain (18). This strain produced (beta)-galactosidase only when acetohydroxybutyrate or acetolactate were present. This allowed the isolation of mutant derivatives that no longer required the inducers in order to produce (beta)-galactosidase. These derivatives produced (beta)-galactosidase even under repressing conditions. When a wild-type ilvC gene was introduced into the derivatives, acetohydroxyacid isomeroreductase was produced constitutively, showing that these mutations were trans-acting. Cis-trans analysis demonstrated that the mutations were trans-dominant. The trans-dominance of these mutations, and the fact that they allowed the strain to express the ilvC promoter constitutively, provides strong in vivo support for the model of positive control of ilvC. Cloning of DNA carrying the mutations into pBR322 and analysis with restriction endonucleases revealed that the mutations were within the ilvY gene. An S-30 extract from ilvY2208 was able to allow induction of (beta)-galactosidase production in vitro from a plasmid carrying an ilvC-lac fusion. With optimal amounts of template and S-30 extract from an ilvY deletion strain, enough (nu) could be produced in vitro to allow induction of the ilvC-lac fusion. The time required for a detectable amount of (beta)-galactosidase to be formed doubled if (nu) was not added to the system. Labelling of the protein products followed by separation on an SDS-polyacrylamide gel demonstrated that (nu) was being made in vitro. With this technique, it was shown that both acetohydroxybutyrate and ppGpp were required for maximal expression of ilvC. ilvY gene expression was also shown not to be under autogenous control. These results suggested that ilvC and ilvY have separate control regions. By uncoupling transcription from translation in vitro, it was shown that ppGpp was required for maximal transcription of ivlC. DNA base sequence determination suggests that the ilvC promoter and the ilvY terminator may both lie within a 250 base pair EcoRI-BglII region, and that ilvY is transcribed from ilvA towards ilvC.

Degree

Ph.D.

Subject Area

Microbiology

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