Purification and characterization of the DNA binding domain of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80.
Ndt80 is a Saccharomyces cerevisiae meiosis-specific transcription factor responsible for promoting the stage-specific expression of a family of genes referred to as middle sporulation genes. Many members of this gene family are essential for the completion of meiotic chromosome segregation. Thus, Ndt80 is essential for the completion of meiosis. Ndt80 is highly regulated both transcriptionally and post-translationally. To facilitate biochemical analysis of Ndt80, we have expressed the DNA binding domain in Escherichia coli and purified the recombinant protein with an affinity chromatography procedure. In addition we have dissected the amino-terminus of Ndt80 to delimit the functional DNA binding domain. This analysis shows that the amino-terminal 40 amino-acids of Ndt80, although not essential for its DNA binding activity, do have an effect on its ability to bind specifically to its target DNA sequence. In addition, we show that the Ndt80 DNA binding domain can be phosphorylated by the meiosis-specific protein kinase Ime2 in vitro, but contrary to our initial hypothesis this phosphorylation does not significantly affect the affinity of Ndt80 for its target DNA sequence.
Sopko
Richelle
R
Department of Biochemistry, University of Alberta, 561 Medical Sciences Building, Edmonton, Alta., Canada T6G 2H7.
Stuart
David T
DT
D000595:Amino Acid Sequence; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D008540:Meiosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C097071:NDT80 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D011494:Protein Kinases; C064979:IME2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases