In vivo analysis of functional regions within yeast Rap1p.
We have analyzed the in vivo importance of different regions of Rap1p, a yeast transcriptional regulator and telomere binding protein. A yeast strain (SCR101) containing a regulatable RAP1 gene was used to test functional complementation by a range of Rap1p derivatives. These experiments demonstrated that the C terminus of the protein, containing the putative transcriptional activation domain and the regions involved in silencing and telomere function, is not absolutely essential for cell growth, a result confirmed by sporulation of a diploid strain containing a C terminal deletion derivative of RAP1. Northern analysis with cells that expressed Rap1p lacking the transcriptional activation domain revealed that this region is important for the expression of only a subset of Rap1p-activated genes. The one essential region within Rap1p is the DNA binding domain. We have investigated the possibility that this region has additional functions. It contains two Myb-like subdomains separated by a linker region. Individual point mutations in the linker region had no effect on Rap1p function, although deletion of the region abolished cell growth. The second Myb-like subdomain contains a large unstructured loop of unknown function. Domain swap experiments with combinations of elements from DNA binding domains of Rap1p homologues from different yeasts revealed that major changes can be made to the amino acid composition of this region without affecting Rap1p function.
Graham
I R
IR
Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.
Haw
R A
RA
Spink
K G
KG
Halden
K A
KA
Chambers
A
A
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007716:Kluyveromyces / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales / Q000235:genetics* / Q000254:growth & development; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C440829:RAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D014157:Transcription Factors