In Vivo Analysis of Functional Regions within Yeast Rap1p

Abstract

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.

ACKNOWLEDGMENTS

This work was supported by a Project Grant from the BBSRC (U.K.) and by the University of Nottingham Research Opportunities Fund. K.A.H. is a M.Phil. student funded by the European Union Social Fund.

We thank Paula Gonçalves, Willem Mager, and Rudi Planta (Amsterdam) for generously providing yeast strain SCR101; Judith Berman (Minnesota) for the anti-Rap1p antibody; Sue Miles for technical assistance; Stuart Ingleston for help with figures; and Paul Sharp for advice on protein alignments.