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|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|
|Doctoral Committee Chair(s):||Gumport, Richard I.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|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.
|Rights Information:||Copyright 1989 Burton, William Seymour|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI8924780|