EXPRESSION AND REGULATION OF THE YEAST TRP5 GENE

WILLIAM SCOTT MOYE-ROWLEY, Purdue University

Abstract

The TRP5 gene is under the general amino acid control system of yeast. The expression and regulation of TRP5 has been analyzed via the use of translational gene fusions between the tryptophan synthase coding region and Escherichia coli 'lacZ. In order to localize the regions of DNA in the 5' flanking sequences important in regulation and expression, a deletion analysis was carried out. These deletions localized the entire control region as being contained within 188 bp upstream from the transcription start site. Further deletion mutants allowed the dissection of the 188 bp region into upstream and downstream halves. Each of the two regulatory regions contains one copy of the TGACT repeat sequence, required for general amino acid control. An additional approach was employed to extend the analysis of sequences important in control of TRP5 which involved the use of synthetic oligonucleotides containing TGACT repeats. The oligonucleotides were inserted into deletion mutants and their ability to restore the function lost in the mutant was assayed by the effect on the downstream TRP5-lacZ fusion. These replacements identified the upstream TRP5 TGACT sequence at -185 as being important in expression and regulation while suggesting the downstream repeat is primarily involved in derepressed expression. The region between these two repeats appears to be important in maintenance of high level basal expression. The role of the TRP5 TATA box region in formation of 5' mRNA termini was also investigated. Northern blot analysis confirmed that steady state TRP5 mRNA levels increased upon general control derepression. A start site at +26 appeared to be preferentially utilized upon derepression. The TATA box was found to be essential for transcription and sequence information downstream from the TATA region appears to be important in determining the start site.

Degree

Ph.D.

Subject Area

Biochemistry

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