The Structural Biology Research Of S.cerevisiae. Histone Chaperone Rtt106 And Human Protein Chaperone NAC

The basic genetic material of life is DNA. In the eukaryotic cells, DNA is packaged with some proteins to form a compact chromosome. These proteins helping DNA package are called histones and the basic element that formed by DNA and histones is called nucleosome. Histone chaperone is a class of proteins that help histone to correctly translocate and perform their functions during their biogenesis, function and metabolism. Rtt106, a histone chaperone from Saccharomyces cerevisiae, binds histone sub-complex H3-H4, and assists the assembly of nucleosome during replication and transcription. Rtt106 also plays crucial roles in the process of heterochromatin formation.In this work, we determined the crystal structure of the core part of Rtt106 at 2.5A resolution, and preliminary studied the mechanism of its function. The core part of Rtt106 (Rtt106-M) is composed of two tightly combined tandom pleckstrin homology (PH) domains. We defined a loop region on the second PH domain of Rtt106-M as the main responsible region for Rtt106 combination with histone H3-H4 sub-complex. Moreover, we identified that Rtt106 has a nucleic acid binding activity and found the main nucleic acid binding region on Rtt106. We proved that the histone-and nucleic acid-binding activities of Rtt106 are indispensible for the heterochromatin formation in yeast using the URA3 silencing assay. Through immunofluorescence assay and chromatin immunoprecipitation assay, we observed and measured the effects of Rtt106's histone-and nucleic acid-binding deficiencies on the distribution of the heterochromatin component protein Sir2 on the telomeric heterochromatin region.Ribosome is the machine where protein synthesis occurs. It reads the coding information from RNA and translates it to the sequence of amino acids composing the protein. Newly synthesized proteins exit the ribosome as unfolded polypeptide chains that must achieve a specific three-dimensional structure to become functionally active under the assistance of a series of proteins and protein complexes. These molecules that assist the correct folding and targeting of polypeptides are called protein molecular chaperones. Nascent polypeptide-associated complex (NAC) binds ribosome at a 1:1 stoichiometry, and is the first molecular chaperone contacting the nascent polypeptide. NAC helps the newly synthesized proteins to fold and target to endoplasmic reticulum and mitochondria correctly. Human NAC is a heterodimer composed of a and 6 subunits. The a subunit alone can forms homodimer and functions as a transcription coactivator in the nucleus.We have determined the crystal structure of the NAC domain of human NAC, which is mainly responsible for the dimerization of its two subunits. One the basis of the structure, we found a novel nucleic acid-binding region on the a subunit of NAC (NACA). This nucleic acid-binding region is exposed in the homodimer of NACA, and has an ability to bind nucleic acid stably; whereas in the heterodimer of a andβsubunits (BTF3), this region is covered by a helix region of BTF3, and loses its activity to bind nucleic acid. Through the immunofluorescence assay in vivo, we discovered that this nucleic acid-binding region is crucial for the location of the NACA homodimer in the nucleus. These results help us find a mechanism of NAC subunits to regulate their distributions and functions in cell by forming different dimers. NAC proteins form homo-and heterodimers to decide their functions as protein chaperone or transcription coactivator, and make a connection between translation and transcription processes in cell.