Functional Analysis Of C-terminal Of The Class Ⅱ Polypeptide Release Factor In Euplotes Octocarinatus

Protein synthesis is one of the most important processes in organism, which includes the processes of initiation, elongation and termination. The processes require two classes of polypeptide release factors. In eukaryotes, in vitro reconstitution of translation termination revealed the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptides. Class I release factors recognize stop codons and hydrolyze ester-bond of aminoacyl-tRNA. The Class II release factors eRF3 is a ribosome-dependent and eRF1-dependent GTPase. Two classes of polypeptide release factors form complex out of ribosome. In the progress of nascent polypeptides release, GTP hydrolysis by eRF3 promotes the function of eRFl on the ribosome.Euplotes octocarinatus is a unicellular protozoa. In Euplotes, UGA encodes cysteine in addition to the conventional cysteine codons, hinting some speciality of the termination of protein synthesis process in the organisms. In E. octocarinatus, two kinds of class I release factors, eRF1a and eRF1b, and one eRF3 have been reported. The E. octocarinatus eRF1b gene and eRF3 gene possesses three UGA stop codons encoding cysteine, respectively, in contrast with no stop codons reassigned in eRF1a gene.In this work, the interaction regions of two classes of polypeptide release factors in Euplotes octocarinatus were analyzed. The following results were obtained.The interaction of two classes of release factors in E. octocarinatus was analyzed by using the in vivo yeast two-hybrid system and in vitro pull down assay. In the two-hybrid analysis, S. cerevisiae strain AH109 was co- transformed with pGBKT7-eRF1a and pGADT7-eRF3. Transformants were analyzed byβ-galactosidase activity. The results revealed two classes of release factors in E. octocarinatus could interact in vivo; In pull down assay, the eRF1a and eRF3 gene were cloned into the vector pRSETC and pGEX-6P-1, respectively. The fusion protein GST-eRF3 was expressed in E. coli BL21 (DE3) and purified by affinity chromatography. The GST-eRF3 fusion protein was detected by Western blotting analysis. The eRF3, after enzymatic cleavage of GST-tail, was obtained. Supernatant containing recombinant His-eRF1a was loaded into the GS4B beads that contain immobilized GST-eRF3.After incubation at 4℃, beads were washed to remove non-specific proteins.Bound proteins were eluted by adding of excess glutathione and analyzed by Western blotting using anti-His antibody. The results indicated two classes of release factors in E. octocarinatus could interact in vitro.Furthermore, in order to identify the eRF1a-binding sites on eRF3 in E. octocarinatus, the eRF3 gene was mutated and eight mutants of eRF3 were obtained. Systematic analysis of the interaction between E. octocarinatus eRF1a and these eRF3 mutants was performed by employing both in vivo yeast two-hybrid assay and in vitro pull down assay. The results revealed: 1) A short portion of the C-terminal domain of eRF3 is sufficient for eRF1a binding in E. octocarinatus. 2) The eRF1a-binding sites on eRF3 are located at a region containing amino acid residues 640-723 in E. octocarinatus eRF3. Amino acid sequence analysis of eRF3 from E. octocarinatus, human, and yeast shows that the eRFla binding domain on E. octocarinatus eRF3 is more similar to that of yeast eRF3, but different from that of human eRF3.On the basis of above results, the eRF3C gene including G domain and eRFla binding domain is cloned into the expression vector pGEX-6P-1. The fusion protein GST-eRF3C was expressed in E. coli BL21 (DE3) and purified by affinity chromatography. The GST-eRF3C fusion protein was detected by Western blotting analysis. The eRF3C, after enzymatic cleavage of GST-tail, exhibited a molecular weight of about 48 KDa. About 2.5 mg pure protein was obtained from per liter of bacterial culture. The fluorescence spectroscopy of eRF3C showed on the excitation at 280 nm and 295 nm, the maximal emission is at 334 nm. Circular dichroism spectroscopy of eRF3C exhibited two negative peaks at 210nm and 220 nm. Second structure calculations indicated 24.0%α-helix, 15%β-sheet, 27.0 % turn and 34.0 % random coli in eRF3C.The Class II release factors are GTPase and can bind GTP and hydrolyze GTP. The activity of eRF3C binding GTP and GDP in E. octocarinatus is studied by employed the fluorescence spectroscopy and circular dichroism spectroscopy. The results showed the E. octocarinatus eRF3C could bind GTP and GDP in the presence of 2 mM MgCl₂, and the binding could induce conformation change of eRF3C. GTP hydrolysis by eRF3C was measured by reverse high performance liquid chromatography. The results showed the E. octocarinatus eRF3C could hydrolyze GTP and it's activity depend on ribosome and eRF1a. The data were analyzed byusing Lineweaver-Burk plots, the results indicated: V_max=4.3 pmol s^-1, K_m = 0.5 mM, K_cat=8.6×10^-2s^-1.