| dc.description | FACT is a highly conserved protein complex that assists in overcoming the repressive effects of chromatin for gene transcription, DNA replication, and DNA repair; however, the exact mechanisms of its actions during these processes remain unclear. FACT is found in all eukaryotes; in the budding yeast Saccharomyces cerevisiae, FACT is composed of two proteins: Spt16 and Pob3. My research has focused on the transcriptional involvement of the Spt16 subunit of yeast FACT, concentrating primarily on two point-mutant forms of this protein: Spt16-E857K and Spt16-E763G. These two mutations were identified by the same transcription-related dominant Spt phenotype, and affect the same region of the Spt16 polypeptide. To further understand how FACT functions in transcription, and more specifically in transcription elongation, I have identified other proteins that interact functionally with these mutant versions of Spt16. These genetic studies, in addition to my biochemical investigations, indicate that these Spt16 mutants affect FACT function in substantially different ways. My results suggest that the spt16-E857K mutant allele encodes a version of Spt16 protein that is impaired for its interactions with nucleosomes and/or nucleosome components, affecting the ability to reassemble nucleosomes following RNA polymerase passage. While spt16-E857K has a spectrum of genetic interactions that primarily involve gene deletions eliminating proteins that function in transcription, the same cannot be said about spt16-E763G, indicating that these two mutations affect different Spt16 protein functions. Perhaps the spt16-E763G mutation causes an impaired interaction between the Spt16 protein and some other protein involved in transcription, rather than a direct nucleosome reassembly defect, as suggested for Spt16-E857K. My findings further the understanding of the functional partners of FACT during transcription, and also indicate that FACT may participate in several different interactions to mediate transcription, as not all Spt16 mutant proteins demonstrate the same pattern of genetic interactions. | en_US |
