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|Title:||A Genetic and Biochemical Analysis of Attenuation Regulation in the Threonine Operon of Escherichia Coli-K12|
|Author(s):||Lynn, Steven Paul|
|Doctoral Committee Chair(s):||Gardner, Jeffrey F.|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Threonine biosynthesis in Escherichia coli K-12 was proposed to be regulated by an attenuation mechanism. A genetic and biochemical analysis was performed to provide more evidence that attenuation was the mechanism utilized to regulate threonine biosynthesis.
The limits of the thr operon regulatory region were defined by cloning the regulatory region in plasmid vectors, analyzing the affect these plasmids had on expression of the chromosomal thr operon, and characterization of the in vitro RNA transcription pattern generated from these templates. A 162 nucleotide transcript was produced when DNA templates extended to the BstEII site upstream of the thrA gene with transcription termination occurring at a rho-independent terminator with 90 percent efficiency.
To characterize the thr operon attenuator, mutants were isolated which affected the RNA helix and thymidine tract. Using a threonine autotroph, generated by a secondary-site $\lambda$ lysogen, lysogenic threonine prototrophs were selected. Most of the mutants identified affected the thr operon attenuator RNA helix, and increased thr operon expression 2-5 fold in vivo. All of the mutants decreased the calculated thermodynamic stability of the RNA secondary structure and most decreased termination at the thr attenuator in vitro. No direct correlation between in vitro termination efficiency and RNA secondary structure could be identified. Removal of less than three of nine thymidines from the thr attenuator did not affect transcription in vitro. Two or less thymidine residues in the template were completely defective in termination in vitro. Expression of galactokinase in vivo from plasmids containing these deletions paralleled the in vitro results.
Transcriptional pausing was observed near position 110 and was enhanced when NusA protein was included. No pausing at the thr attenuator was observed when the thymidine tract was deleted from the template.
The specificity of the attenuation response was analyzed by changing the threonine and isoleucine codons in the leader RNA to histidine and tyrosine by oligonucleotide-directed mutagenesis. In this mutant, thr operon expression was sensitive to the hisT allele but not to a temperature-sensitive thrS allele, the opposite response of the wildtype. The leader peptide was identified using a cell-free transcription/translation system.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
|Date Available in IDEALS:||2014-12-16|