This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Part of my research focuses on a crucial RNA processing and degradation machinery, the exosome, which has often been called the proteasome for RNA. Initially identified from mutations causing 5.8S rRNA 3 end processing defects, the exosome is a conserved 300 - 400 kD 3 -to-5 exoribonuclease complex present in both the nucleus and the cytoplasm of eukaryotic cells. The exosome consists of a core of ten proteins: Rrp4p, Rrp40p to Rrp46p, Mtr3p, and Csl4p (yeast nomenclature), all are essential for cell viability. Six of them are phosphorolytic RNases; the other four are predicted to be hydrolytic RNases. In the nucleus, the exosome is required for the 3 end formation of 5.8S rRNA and the degradation of the 5 -external transcribed spacer; participated in the 3 end maturation of small nuclear and nucleolar RNAs; and involved in degradation of inefficiently spliced or hypoadenylated pre-mRNAs. The cytoplasmic exosome is involved in the degradation of mRNAs containing premature termination codons, lacking termination codons, or bearing AU-rich elements (AREs) near the 3 untranslated region. To gain insights into the architecture and enzymatic mechanism of the exosome, I have proposed to determine the crystal structures of the 300 kD, 4-preotein archaeal exosome, the 10-protein eukaryotic exosome, and the exosome-RNA substrate complexes. The long term goals include determination of exosome-adaptor protein complexes to investigate its regulation. Exciting progress has been made in expression, purification, and crystallization of the archaeal exosome complex. Well-behaving crystals diffracted X-ray to ~ 2.4 resolution at synchrotron radiation source. Additional beam time is needed to complete Se-MAD phasing and carry out structural enzymology studies on the archaeal exosome complex. The second part of my research focuses on Signal Recognition Particle (SRP) mediated co-translational translocation of proteins across or into membranes. This vital cellular process requires the translating ribosome to be membrane-targeted by the SRP, a ribonucleoprotein complex conserved in all three kingdoms of life. SRP recognizes the hydrophobic signal sequence of the nascent protein emerging from the ribosome, resulting in transient elongation arrest in eukaryotes, and targets the ribosome to the membrane via a GTP-dependent interaction with the SRP receptor (SR). The ribosome is then handed over to the translocon, where protein translation and translocation happens simultaneously. The SRP-SR dissociates following GTP hydrolysis and SRP cycle resumes. Crystals of the E. coli SRP bound to the receptor complex has been obtained and diffracted X-ray to medium resolution of 6 . Synchrotron radiation source is absolutely needed to screen for cryoprotection conditions and alternative crystal forms to determine the complex structure. Future research will also target the more complicated eukaryotic SRP complex to fully understand the role of RNA in the co-translational protein targeting process.
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