A new method for the construction of a mutant library with a predictable occurrence rate using Poisson distribution.
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.
Seong
Ki Moon
KM
Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Park
Hweon
H
Kim
Seong Jung
SJ
Ha
Hyo Nam
HN
Lee
Jae Yung
JY
Kim
Joon
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids; D017354:Point Mutation; D016012:Poisson Distribution; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors