| Summary: | 碩士 === 國立陽明大學 === 遺傳學研究所 === 94 === The RSC complex, which consists of at least fifteen proteins, alters the histone-DNA interactions and nucleosome mobility by ATP hydrolysis, allowing transcription factors accessing to chromatin for a broad range of gene regulation in yeast genome. Htl1p is a component of RSC complex. In the absence of Htl1p, the integrity of RSC complex is disturbed. To better understand the role of Htl1p in the RSC complex, here we examine the components of RSC complex by tandem affinity purification and compare the differences of RSC components purified from RSC2-TAP, which chromosome RSC2 gene was fused with a TAP-tag at the c-terminus and its htl1 deletion derivative RH2-TAP. We observed that the Rsc58p seems to be unstable in RH2-TAP. Moreover, in the presence of Htl1p mutant protein (Htl1-7), the protein profile is different from that of RSC2-TAP and RH2-TAP. By immunoblot analysis, we observed that the relative quantity of Rsc58p and Rsc8p in the presence of Htl1-7 protein is decreased in the purified protein profile. Htl1p and Rsc58p associated proteins were also purified by tandem affinity purification. They are essential RSC components. However, in comparison of the protein profiles purified by the rsc8 mutants derived from RSC2-TAP and RH2-TAP, there’s no difference among them. Our preliminary data showed that deletion of htl1 affects the interaction between Rsc8p and RSC complex. To further understand the interaction between Rsc8p and RSC complex, we design a fusion protein SLR, using a linker containing the GGMP repeats of Hsc70 to connect Sth1p and Rsc8p. Yeast cells contain SLR with deletion of htl1 failed to survive. But SLR could rescue the lethal phenotype caused by down-regulation of RSC8 expression in tet-off system, indicating that SLR may replace the function of both RSC8 and STH1. Overall speaking, above results further confirm that Htl1p may be crucial for the stability of
RSC complex.
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