A new method for the construction of a mutant library with a predictable occurrence rate using Poisson distribution

Ki Moon Seong, Hweon Park, Seong Jung Kim, Hyo Nam Ha, Jae Yung Lee, Joon Kim

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

A yeast transcriptional activator, Gcn4p, induces the expression of genes that are involved in amino acid and purine biosynthetic pathways under amino acid starvation. Gcn4p has an acidic activation domain in the central region and a bZIP domain in the C-terminus that is divided into the DNA-binding motif and dimerization leucine zipper motif. In order to identify amino acids in the DNA-binding motif of Gcn4p which are involved in transcriptional activation, we constructed mutant libraries in the DNA-binding motif through an innovative application of random mutagenesis. Mutant library made by oligonucleotides which were mutated randomly using the Poisson distribution showed that the actual mutation frequency was in good agreement with expected values. This method could save the time and effort to create a mutant library with a predictable mutation frequency. Based on the studies using the mutant libraries constructed by the new method, the specific residues of the DNA-binding domain in Gcn4p appear to be involved in the transcriptional activities on a conserved binding site.

Original languageEnglish
Pages (from-to)442-450
Number of pages9
JournalJournal of Microbiological Methods
Volume69
Issue number3
DOIs
Publication statusPublished - 2007 Jun

Keywords

  • Gcn4p
  • Poisson distribution
  • Random mutagenesis
  • bZIP transcriptional factor

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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