An analysis of the effect of mutations in the leader region of the threonine operon of Escherichia coli on attenuation or transcription termination
Scott, Harlan Bernard, II
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https://hdl.handle.net/2142/19344
Description
Title
An analysis of the effect of mutations in the leader region of the threonine operon of Escherichia coli on attenuation or transcription termination
Author(s)
Scott, Harlan Bernard, II
Issue Date
1991
Doctoral Committee Chair(s)
Gardner, Jeffrey F.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Language
eng
Abstract
The expression of the threonine operon of Escherichia coli is controlled by attenuation. An open translational reading frame in the leader region preceding the structural genes contains 21 codons with eight threonine and four isoleucine codons. The model for attenuation proposes that regulation depends on the translation of the 21 amino acid leader peptide. Oligonucleotide-directed mutagenesis was used to introduce mutations into the leader region. Thirty-nine mutants were isolated. The effects of individual mutations on regulation were studied by making fusions to the lacZ gene, reducing the plasmid copy number, and assaying for the $\beta$-galactosidase activity.
Five mutants with two frameshift mutations each were constructed. The double frameshift mutants changed the leader peptide in two ways. First, the mutants had differing numbers of threonine and isoleucine codons. Second, the mutants had the threonine and isoleucine codons changed to differing numbers of histidine codons. Changing three threonine and one isoleucine codons abolished regulation by excess threonine and isoleucine. The presence of two histidine codons derepressed the operon in a hisT::Km background. Three mutations that would be predicted to block the translation of the normal leader peptide were constructed. All three mutants showed loss of regulation.
The attenuator in the leader region encodes a rho-independent terminator. The terminator consists of a stem-loop structure with six bases in the loop and eight basepairs in the stem. The stem-loop is followed by nine rU residues and preceded by six rA residues. Various mutations in the threonine attenuator were assayed for their effects on transcription termination efficiency. As the number of rU residues in the terminator decreased from eight to one, the termination efficiency decreased. As the number of rA residues preceding the stem decreased from eight to one, the termination efficiency slightly increased. The sequence of the loop was also found to have a slight affect on termination efficiency. The sequences distal to the rU residues, which were non-transcribed, also had a slight affect on termination efficiency. Finally, a combination of changes in the stem with a change either in the loop or sequences distal to the rU residues had an affect on termination efficiency.
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