In vivo analysis of functional regions within yeast Rap1p

We have analyzed the in vivo importance of different regions of Rap1p, a ye ast transcriptional regulator and telomere binding protein. A yeast strain (SCR101) containing a regulatable RAP1 gene was used to test functional com plementation by a range of Rap1p derivatives. These experiments demonstrate d that the C terminus of the protein, containing the putative transcription al activation domain and the regions involved in silencing and telomere fun ction, is not absolutely essential for cell growth, a result confirmed by s porulation of a diploid strain containing a C terminal deletion derivative of RAP1. Northern analysis with cells that expressed Rap1p lacking the tran scriptional activation domain revealed that this region is important for th e expression of only a subset of Rap1p-activated genes. The one essential r egion within Rap1p is the DNA binding domain. We have investigated the poss ibility 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 re gion abolished cell growth. The second Myb-like subdomain contains a large unstructured loop of unknown function. Domain swap experiments with combina tions of elements from DNA binding domains of Rap1p homologues from differe nt yeasts revealed that major changes can be made to the amino acid composi tion of this region without affecting Rap1p function.