Cloning, Expression, Purification And Preliminary Crystallographic Analysis Of Pub1-RRM2 And RPA14 From Saccharomyces Cerevisiae

一,Cloning, expressing, purification, crystallization and preliminary X-ray diffraction analysis of the second RRM of Pub1 from Saccharomyces cerevisiaIn eukaryotes,gene expression is a highly regulated process which involves regulation at both transcriptional and post-transcriptional levels. mRNA turnover, which is mediated by the interplay between the different cis-element sequences localized in the target mRNAs and the various trans-acting factors interacting with them, plays an important role in post-transcriptional regulation. RNA binding protein is an abundant protein fond in eukaryotes, which plays an important role in diverse biological processes by specifically binding different RNAs and DANs. RNA binding protein always contains different domains, while the RNA recognition motif (RRM) is the most abundant protein motif found in many RNA-binding proteins. RRM can bind to specific DNA or RNA sequences to regulate their stabilities and is also involved in protein-protein interaction.Poly(U)-binding protein 1 (Pub1) is an important polyadenylated RNA-binding protein in Saccharomyces cerevisia. It is detected both in nuclear and cytoplasm and there is little difference in the distribution patterns between the nuclear and cytoplasm. PUB1 binds not only to poly(A) but also to poly(G) and poly(U), specifically has high affinity to poly(U). Pub1 is involved in the regulation of mRNA turnover as a trans-acting factor. It can bind to the AU-rich elements (AREs) and stabilizer elements (STEs)-containing transcripts to protect them from degradation. In addition, Pub1 is also suggested to be involved in mRNA processing and export. Pub1 contains three RRMs, which each RRM contains the conserved box RNP1 and RNP1, play important roles in binding to AREs and STEs to regulate their stabilities. The three RRMs have different functions, while the second RRM may play a central role in poly(U) binding and determine the binding specificity of Pub1.The DNA coding sequence for the second RRM of yeast Pub1 was amplified from Saccharomyces cerevisiae genome by PCR and the recombinant plasmid RRM2-pET22b was constructed. The recombinant protein with a–LEHHHHHH tag at the C-terminus was expressed highly as a soluble protein in E. coli Rosetta (DE3). The target protein which turned out to be about 95% pure was obtained after purified by Ni-NTA column. The protein exists as a monomer in solution by size exclusion chromatography assay and no obvious degradation was observed in SDS-PAGE. Crystallization experiments were performed using the hanging-drop vapor diffusion method. After several rounds of optimization, a well diffracting single crystal was obtained and a complete diffraction data set to 1.69 ? resolution of was collected. The crystal belongs to space group H3 with unit-cell parameters a = b = 73.11?, c = 41.1 ?. Both Matthews coefficient analysis and the self-rotation function suggested the presence of one molecule per asymmetric unit in the crystal. The molecular-replacement method was performed using the structure of the second RRM of human protein TIA-1 as a search model. The further refinement of the structure is on the way.二,Cloning, expression, purification and the structural research of RPA14There are three distinct types of eukaryotic nuclear RNA polymerases: RNA polymerase (pol)Ⅰ, polⅡ, polⅢ. Saccharomyces cerevisiae polⅠconsists of 14 subunits. The core structure contains 10 subunits. In fact, polⅠrequires four specific subunits for specific transcription of rDNA, that is A49, A43, A34.5, A14. A43 forms a heterodimer with A14 which is conserved in a variety of eukaryotes. A14/A43 also interacts with an rDNA-specific transcription factor (Rrn3p) for polⅠrecruitment to the rDNA promoter and is able to bind to single-stranded RNA. A yeast strain where the RPA14 gene is disrupted is viable, but the purified RNA polⅠwas found to be inactive in transcription. It is suggested that RPA14 plays an important role in polⅠ. Moreover, RPA14 can stabilize the interaction between A43 and the core enzyme.The gene that codes for RPA14 was amplified from Saccharomyces cerevisiae genome and the protein is overexpressed in Escherichia coli strain BL21 (DE3). The protein is difficultly purified. The protein was about 90% pure after purified by Ni-NTA column, HiloadTM 16/60 SuperdexTM 75 column and DEAE column. Then the protein was using for crystallization experiments, but there was no crystals obtained.