Preliminary Structural Studies Of RsmA From Staphylococcus Aureus And ISW2 Chromatin Remodeling Complexes From Saccharomyces Cerevisiae

1. Preliminary crystallographic study of RsmA from Staphylococcus aureusStaphylococcus aureus is an important human pathogen which can cause purulent infection and other diseases. One way to inhibit Staphylococcus aureus infection is to inhibit the expression of toxic proteins and their associated toxic molecules. RsmA protein is a ribosomal RNA small subunit methyltransferase and it can catalyze the N6 dimethylates of two adjacent adenosines (A1518 and A1519 in Escherichia coli) in the loop of a conserved hairpin near the 3’-end of 16S rRNA in the 30S particle. Previous study has revealed that loss of RsmA could profoundly impair ribosome biogenesis and be deleterious for cell growth.For this part study, we want to explore the structural basis of RsmA protein catalyzing N6 dimethylates of two adjacent adenosines and indentify potential possible antimicrobial drug targets via analysis of RsmA structure. So we cloned, expressed and crystallized the native RsmA protein and SeMet-RsmA protein, then obtained a set of X-ray data with the resolution 3.2 A using X-ray diffraction. We tested the binding ability of RsmA with SAM and RsmA with SAH by ITC assay, then co-crystallized RsmA with SAM and SAH, and obtained the RsmA-SAH, RsmA-SAM complex crystals. The quality of crystals is not well to be further optimized.2. Structural exploring of ISW2 chromatin remodeling- complexes from saccharomyces cerevisiaeThe chromatin exists in the form of nucleosome in eukarytic cells and the packaging structure is very close, so that transcriptional regulatory proteins can’t bind to the DNA and this can influence gene expression. ATP-dependent chromatin remodeling complexes can use the energy of ATP hydrolysis to change the status of chromatin and regulate gene expression through moving, assembling and replacing nucleosomes. There is a conserved ATP-dependent chromatin remodeling complexes family called ISWI family in Saccharomyces cerevisiae, which contains ISW1 and ISW2 chromatin remodeling complexes. ISW2 chromatin remodeling complexes contains Isw2, Itcl, Dpb4 and Dlsl four subunits and can regulate the spacing of nucleosomes to remodel the chromatin. Compared to the ISWI, the length of DNA between nucleosomes is not required and the structure of nucleosome is not destroyed in the process of chromatin remodeling by ISW2.In this part work, we try to resolve the ISW2 complex and subunits structure to explore the mechanism of remolding. Each subunit of complex was cloned-, expressed and crystallized.We obtained the Isw2 crystals but the diffraction effect is not good. We explored the DNA binding ability of Isw2 and Dpb4+Dlsl by ESMA experiment and obtained the Dpb4+ Dlsl+ dsDNA complex crystals by adding dsDNA into Dbp4+Dlsl complex. We have mapped the fragments of Isw2 that interact with Dpb4+Dlsl through pull down and the complex of Isw2 fragments, Dpb4 and Dls1 are being crystallized to obtain the Isw2+Dpb4+Dls1 crystals. At the same time, the Isw2 and Dpb4+Dlsl+ dsDNA crystals are optimized and hope to get the structure information.