| Background:Cren7is a newly discovered conservative, non-specific chromatin proteinin the Crenarchaeota. Chromatin proteins play an important role for genome packaging andgene regulation in prokaryotic, archaea and eukaryotes, But the understanding of Archaealchromatin proteins is still in its infancy. There is no uniform chromatin structure for Archaeal,Crenarchaeota as a major branch of archaea, the discovery and research of Cren7will greatlyenrich the genetics of archaea.The application of the X-ray crystallography on the structuralinformation of biological macromolecules also make the crystallography becomes an importantmeans to analyze the structure and function of Cren7, co-crystallize Cren7and DNA, select thecrystal that is suitable for diffraction.Aim:Cren7as a newly discovered chromatin proteins in the Crenarchaeota, themechanism that Crenarchaeota genome is compressed and packed by Cren7is still unkown.This study will combine Cren7with long-chain DNA and the DNA base mismatch, and selectthe two key sites of point mutations of Cren7respectively, then combined with the syntheticDNA fragment. Collect the X-ray diffraction data, provide the basis for revealing thepackaging mechanism that Crenarchaeota genome is compressed and packed by Cren7..Methods:The gene of Cren7was cloned from Sulfolobus shibatae, after inducedexpression and protein purification, respectively combine with long-chain DNA fragments andDNA fragments of base mismatches; then according to the crystal structure of Cren7and theDNA complexes, make a point mutation of the two key amino acids(28L,51R)which combinesDNA, Point mutations Cren7were combined with8bpDNA fragments, then crystal theseCren7-DNA complexes, after a lot of screening and optimization, filtering out the crystals thatis appropriate for diffraction.Result:High-resolution crystal of Cren7with long DNA fragmentsã€Cren7with the DNAof mismatch and five mutational Cren7respectively with the8bp DNA fragments except forthe Cren7(R51A)-DNA were got, the X-ray diffraction data of these compound will be used forrevealing the machanism that Crenarchaeal genome is packaged by Cren7through further dataanalysis. |
