IDENTIFICATION OF MUTATIONS IN THE ESCHERICHIA COLI RECA AND LEXA REGULATORY LOCI.

Persistent Link:
http://hdl.handle.net/10150/187848
Title:
IDENTIFICATION OF MUTATIONS IN THE ESCHERICHIA COLI RECA AND LEXA REGULATORY LOCI.
Author:
WERTMAN, KENNETH FRANKLIN.
Issue Date:
1984
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
This report describes the development and use of an expression vector system based on the single-stranded DNA bacteriophage M13. A derivative of M13mp8, designated M13mp8/P, was prepared in which the promoter and N-terminal codons of bacterial genes may be fused to a portion of β-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes, and to determine the mutational changes by dideoxy sequence analysis. The feasibility of this method was demonstrated by identification of a large number of mutations in the regulatory regions of two genes, recA and lexA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. My results suggest that this method will be generally useful for mutational analysis of transcriptional and translational regulatory elements. The mutants that were isolated by the above approach were used to investigate the specificity of LexA protein binding by quantifying the repressibility of a several mutant recA and lexA operator/promoter regions fused to the E. coli galactokinase (galK) gene. The results of this analysis indicated that two sets of four nucleotides (terminal nucleotide contacts), one set at each extreme end of the operator, are most critical for repressor binding. In addition, our results indicate that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator had comparable effects on repressibility. The inferred symmetry of the interaction justified the reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus: (5') CTGTATAT. Although the first four positions of this half-site sequence have the greatest effect on LexA repressor binding, the last four are well conserved among binding sites and appear to modulate repressor affinity. The role of the terminal nucleotide contacts and the mechanism by which the internal sequences affect repressor binding is discussed.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Escherichia coli -- Genetics.; Bacterial genetics.; Genetic regulation.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Molecular and Cellular Biology; Graduate College
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleIDENTIFICATION OF MUTATIONS IN THE ESCHERICHIA COLI RECA AND LEXA REGULATORY LOCI.en_US
dc.creatorWERTMAN, KENNETH FRANKLIN.en_US
dc.contributor.authorWERTMAN, KENNETH FRANKLIN.en_US
dc.date.issued1984en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThis report describes the development and use of an expression vector system based on the single-stranded DNA bacteriophage M13. A derivative of M13mp8, designated M13mp8/P, was prepared in which the promoter and N-terminal codons of bacterial genes may be fused to a portion of β-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes, and to determine the mutational changes by dideoxy sequence analysis. The feasibility of this method was demonstrated by identification of a large number of mutations in the regulatory regions of two genes, recA and lexA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. My results suggest that this method will be generally useful for mutational analysis of transcriptional and translational regulatory elements. The mutants that were isolated by the above approach were used to investigate the specificity of LexA protein binding by quantifying the repressibility of a several mutant recA and lexA operator/promoter regions fused to the E. coli galactokinase (galK) gene. The results of this analysis indicated that two sets of four nucleotides (terminal nucleotide contacts), one set at each extreme end of the operator, are most critical for repressor binding. In addition, our results indicate that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator had comparable effects on repressibility. The inferred symmetry of the interaction justified the reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus: (5') CTGTATAT. Although the first four positions of this half-site sequence have the greatest effect on LexA repressor binding, the last four are well conserved among binding sites and appear to modulate repressor affinity. The role of the terminal nucleotide contacts and the mechanism by which the internal sequences affect repressor binding is discussed.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectEscherichia coli -- Genetics.en_US
dc.subjectBacterial genetics.en_US
dc.subjectGenetic regulation.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMolecular and Cellular Biologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.identifier.proquest8504241en_US
dc.identifier.oclc693373504en_US
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