Abstract
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.
Highlights
Pit-1 is a member of the POU family of transcription factors, which contain a bipartite DNA binding domain
Sequence analysis led to the identification of 42 different point mutations within the Pit-1 DNA binding domain, which resulted in altered Pit-1 function in yeast (Table I and Fig. 2)
By using a strategy involving random, in vitro chemical mutagenesis and phenotypic screening in yeast, we have identified a series of point mutations that alter the function of the Pit-1 DNA binding domain (POU domain)
Summary
Pit-1 is a member of the POU family of transcription factors, which contain a bipartite DNA binding domain. 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. The variable length and sequence of the region connecting the POU-specific and the POU homeodomain has led to the suggestion that this region functions as a flexible linker This view has been reinforced by the finding that insertion of six alanine residues into the connecting link of Oct-1 has no effect on DNA binding [13]. This approach has permitted the identification of a number of point mutations, which alter the ability of the Pit-1 POU domain to bind to DNA
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