The activation specificities of wild-type and mutant Gcn4p in vivo can be different from the DNA binding specificities of the corresponding bZip peptides in vitro.
Single amino acid substitutions which previously have been shown to alter the DNA binding specificity of a Gcn4p bZip peptide in vitro were transformed to full length Gcn4p, and activation of a test promoter carrying various palindromic and pseudo-palindromic binding sites was measured. All mutations were found to have different phenotypes, and the first change-of-specificity mutants for Gcn4p in vivo are described. The comparison of plasmids encoding no protein or a particular Gcn4p mutant with broadened activation specificity in gcn4 and gcn4 acr1 genetic backgrounds revealed three new DNA targets of the yeast Acr1p repressor. Surprisingly, we found the activation specificities Gcn4p and the mutants tested in vivo to be generally different from DNA binding specificities of the corresponding bZip peptides in vitro. Especially, the proteins respond differently, in vitro and in vivo, on changes in half site spacing of the DNA binding sites. We present data which largely exclude that the differences between in vivo and in vitro-derived results are due to differences in protein structure, or to the presence of competing protein factors in the yeast cell. We conclude that the differences between in vitro and in vivo-derived results are caused by differences in the degree of flexibility of the target DNA sequences in vitro and in vivo.
Suckow
M
M
Institut für Mikrobiologie der Heinrich-Heine-Universität, Düsseldorf, Germany.
Hollenberg
C P
CP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D051790:G-Box Binding Factors; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D016350:Leucine Zippers / Q000235:genetics; D009154:Mutation; D010641:Phenotype; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D050976:Basic-Leucine Zipper Transcription Factors; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051790:G-Box Binding Factors; D012097:Repressor Proteins; C077832:SKO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D011494:Protein Kinases