A study of the molecular mechanism of the attenuation response in the threonine operon of Escherichia coli by synthesis of mutant attenuators
Burton, William Seymour
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/22367
Description
Title
A study of the molecular mechanism of the attenuation response in the threonine operon of Escherichia coli by synthesis of mutant attenuators
Author(s)
Burton, William Seymour
Issue Date
1989
Doctoral Committee Chair(s)
Gumport, Richard I.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Chemistry, Biochemistry
Language
eng
Abstract
To study the role of the competing terminator and antiterminator RNA structures in attenuation regulation of the thr operon of Escherichia coli, nine related attenuators with altered symmetry were made. Transfersions that destroy or invert a base pair near the middle of the base paired regions of the alternative stem-and-loop structures of the thr regulatory region were created to examine the role of the competing secondary structures in the attenuation response. The variants include the wild type; substitution mutants 106C, 136G and 155C; the three corresponding double mutants, 106C/136G, 106C/155C, and 136G/155C; the triple mutant, 106C/136G/155C; and a single-base deletion at position 106 (106$\Delta$). The frameshift mutation removes the leader translation stop codon to determine whether unstopped ribosomes translating the distal regulatory region interfere with terminator function. All nine variants, including the wild type control, bear a deletion of position 165 as an artifact arising from the gene synthesis ligations.
The mutants were fused to a lacZ sequence and stably integrated into the E. coli chromosome. Consistent with the proposed role for each of the alternative structures, assays of the variants showed that destabilization of the terminator hairpin caused increased expression, while disruption of the antiterminator caused decreased expression. Stability effects in the antiterminator were not observed, however, when the terminator was destabilized. Inverted base pairs functioned well in their respective hairpin structures, and the triple mutant exhibited wild type activity levels. All terminator mutations abolished the regulatory response to amino acid availability; the regulation was restored when the corresponding base change in the antiterminator restabilized the terminator structure. The frameshift mutant has increased expression and cannot regulate expression in response to differences in amino acid levels.
DNA templates bearing the eight substitution mutations were transcribed in a purified RNA polymerase system with the goal of assessing their intrinsic termination efficiencies. In transcription assays performed with GTP, mutational effects were slight and did not correlate with terminator hairpin stability. Transcriptions performed with ITP in place of GTP produced lower termination efficiencies that varied not only with terminator stability, but also with antiterminator stability; the mutational effects paralleled those observed in vivo.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
