Study On A New Domain In Rat Ribosomal RNA And The Deletion Mutation Of Cinnamomin A-chain

This dissertation consists of three parts:Part â… Cleavage-site of eukaryotic ribosomal RNA by a novel ribosome-inactivating proteinFrom the seeds of Biota orientalis was isolated a novel ribosome-inactivating protein (RIP) - orientin that was capable of inactivating rat ribosome and of inhibiting in vitro protein synthesis by a way different from the RNA N-glycosidase. As a specific ribonuclease, orientin cleaved only a single phosphodiester bond between the cytidine residue at position 4451 and the adenosine residue at position 4452 in rat 28S ribosomal RNA and hence produced a RNA fragment that is composed of 333 ribonucleotides at the 3' end of rat 28S ribosomal RNA. The 5'-terminus of the fragment was identified to be a hydroxyl. The sequence around the cleavage-site was highly conserved in certain higher eukaryotes. The domain that contained the cleavage-site by this novel RIP was important for the ribosomal function in protein synthesis. Also this novel RIP could be useful for probing the structure of a new specific functional domain RNA in the ribosome.Part â…¡ Both the N-terminal and C-terminal regions are crucial for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNACinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved Sarcin/Ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. Cinnamomin A-chain can also cleave supercoiled circular double-stranded DNA into the nicked and linear forms. The recombinant cinnamomin A-chain and its four mutants with the N-terminal 52 amino acids residues or/and the C-terminal 51 amino acids residues deleted have been expressed in E coli and purified after refolding. Recombinant cinnamomin A-chain, which exhibited the RNA N-glycosidase activity, could also release adenines from the supercoiled double-stranded DNA, whereas the deletion mutants could deadenylate neither ribosomal RNA nor supercoiled DNA. Additionally, unlike the recombinant cinnamomin A-chain, the deletion mutants could not cleave supercoiled DNA into nicked and linear forms. Taken together, these results revealed that both the N-terminal and the C-terminal regions are required for cinnamomin A-chain to depurinate ribosomal RNA and supercoiled double-strandedDNA, and that the activity to cleave supercoiled DNA is an intrinsic property of cinnamomin A-chain.Part â…¢ Nonspecific deadenylation on Sarcin/Ricin Domain RNA catalyzed by Gelonin under acidic conditionsGelonin is a single-chain ribosome-inactivating protein (RIP) that can hydrolyze the glycosidic bond of a highly conserved adenosine residue in the sarcin/ricin domain (SRD) of the largest RNA in ribosome and thus irreversibly inhibit protein synthesis. Recently, the specificity in substrate recognition was challenged by the fact that gelonin could remove adenines from some other oligoribonucleotide substrates. However, the site-specificity of gelonin to deadenylate various substrates were unknown. Hereby, the effect of pH values upon site-specificity of the deadenylation activity of gelonin was studied using the synthetic oligoribonucleotide (named SRD RNA) that mimicked the rat ribosomal sarcin/ricin domain RNA. Interestingly, gelonin gradually acquired the ability to nonspecifically remove adenines from SRD RNA when pH values changed from neutral to acidic condition. Another two SRD RNA mutants, either with the conserved adenosine deleted or with the tetraloop converted, showed very similar cleavage pattern to wild type SRD RNA, underscoring the important role of pH value in site-specificity of recognition by gelonin. Furthermore, the RNA N-glycosidase activity of gelonin was also enhanced with the decreasing of pH values. In addition, no obvious change was observed in the molecular conformation of gelonin at various pH values. Taken together, our data implied that the protonation of adenosines in SRD RNA was potentially an important factor for the non-specific...