In vivo mutational analysis of the DNA binding domain of the tissue-specific transcription factor, Pit-1.
Pit-1 is a member of the POU family of transcription factors, which contain a bipartite DNA binding domain. The DNA binding domain consists of a POU-specific domain and a POU homeodomain. Each of the subdomains can interact with DNA independently, but both subdomains are required for high affinity, sequence-specific DNA binding. To examine the contributions of individual amino acids to the function of the DNA binding domain of Pit-1, we developed an approach involving random, in vitro mutagenesis followed by functional screening in Saccharomyces cerevisiae. Using this strategy, we identified a number of point mutations that altered the function of the Pit-1 DNA binding domain. Mutations that altered Pit-1 function were found in both the POU-specific and the POU homeodomain. Most of the mutations involve amino acid residues that are conserved in POU factors. One of the more frequent kinds of mutation affected residues located in the hydrophobic core of the protein. Another common mutation involved amino acids that are thought to make specific contacts with DNA. These mutations define a number of amino acid residues that are important for the function of the DNA binding domain of Pit-1.
Liang
J
J
Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland 97201, USA.
Moye-Rowley
S
S
Maurer
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; C494187:POU1F1 protein, human; D050817:Transcription Factor Pit-1; D014157:Transcription Factors