Construction and characterization of oligonucleotide-directed and conventionaltrpR missense mutations

Byron Thomas Hagewood, Purdue University

Abstract

Saturation mutagenesis has been performed on codon 71 of the E. coli trpR gene. These mutations were created by oligonucleotide-directed mutagenesis of a trpR gene fragment within a M13mp cloning vehicle. A Rec-promoted strategy was developed which allowed for positive selection of allele replacement from M13 to chromosomal sequences. This strategy should be applicable to the mutagenesis of many types of proteins. Insufficient homology was postulated as the reason for an inability to successfully recombine the missense mutations onto the chromosome. The CUV107 trpR mutation isolated by Reznikoff and Thornton (1972) was cloned, sequenced and characterized. The CUV107 mutation was found to consist of two nucleotide switches, which would be predicted to result in a single missense mutation, Trp99 $\to$ Arg. A temperature resistant trpPO-lacZ lambda phage was constructed. In vivo $\beta$-galactosidase assays showed that the mutant repressor has a reduced ability to regulate the trp promoter at 30$\sp\circ$C and 42$\sp\circ$C. The mutant protein was overproduced and partially purified. In vitro protection assays showed that the mutant repressor binds trp operator DNA very poorly at 42$\sp\circ$C. Computer analysis indicates that the reduced affinity for trp operator at 30$\sp\circ$C is caused by steric clashes between residue 99 of one monomer and residues 28, 32 and 34 of the other monomer. These steric clashes result in perturbation of the helices of Trp repressor which comprise the HTH-domain.

Degree

Ph.D.

Advisors

Somerville, Purdue University.

Subject Area

Molecular biology|Microbiology|Biochemistry

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