The substrate specificity of monomeric and dimeric forms of c~-sarcin was investigated by membrane blotting procedures. Dimeric ~-sarcin fails to inactivate ribosomes as well as to hydrolyze mini-stem-loop RNA, whereas monomeric Separation ofdimer andmonomer ofoasarcin on PVDFmembraneMonomeric and dimeric a-sarcin were separated by electrophoresis oc-sarcin catalyzes both substrates. Both monomeric and dimeric on a 15% polyacrylamide gel containing SDS and electrophoretically cc-sarcin are effective ribonncleases that are displayed by in situ transferred onto PVDF membrane (Micron Separation Inc., MA, RNA-impregnated gel electrophoresis. The same purine base USA) [18]. The localization of monomers and dimers on the memspecificity was detected for both dimeric and monomeric forms, brane was visualized by Ponccau S staining (Merck Chem. Co.). Di-~-Sarcin is also an effective deoxyribonuclease to supercoiled mers and monomers were cut out from blotted membrane and washed DNA. The action of ~-sarcin as deoxyribonuclease and extensively with distilled water. They were stored for future use. The ribonuclease is inhibited by the presence of SDS (3.5 x 10 6 amounts of protein on the membrane were determined by amino acid M); the inhibition on ribonuclease, but not on deoxyribonuclease, analysis according to previous procedures [19]. The molar ratio of monomer and dimer (7 to 1) on the membrane was quantitatively is reversible if the proteins are renatured, calculated.In an exchange experiment, the electrophoretically blotted PVDF Rabbit reticulocyte ribosomes were used as the substrates. The assay RNA [6][7][8]. The action is specific; only one phosphodiester was done in appropriate amounts of protein for monomer, dimer, and bond in the sarcin domain of 23S 28S rRNA is hydrolyzed native ec-sarcin --1.2, 1.2, and 0.8 × 10 -8 M, respectively, a-fragments generated from large subunit 28S rRNA were scored by a composite [7,8]. It also cleaves a mini-stem-loop RNA that mimics the gel. sarcin domain [9][10][11]. At higher concentrations, the protein can act as a ribonuclease [12]. It carries purine-specific endo- Hydrolyzing mini-stem-loop RNA by membrane-bound ~-sarcinnuclease activity to single-and double-stranded RNA [12,13].The specific ribonucleolytic activities of dimers and monomers were directly examined by their abilities to cleave the mini-stem-loop RNA Besides the ribonuclease activity, a-sarcin has previously been that mimics sarcin domain of rat liver ribosomes. A eDNA template documented to have deoxyribonucleolytic activity [12]. Cleavthat carries T7 promoter and sequences complementary to the sarcin age of supercoiled DNA by ~-sarcin and other related ribodomain of 28S rRNA of rat liver ribosomes was synthesized chemitoxins were reported recently [14].cally using DNA synthesizer (API 470A). The template was further purified by electrophoresis on 20% polyacrylamide gel in TE buffer The dimeric form of a-sarcin is constantly observed and is a (89 mM Tris-borate, pH 8.3; 1 mM EDTA). Radioactive 35-mer un...

The regional structure of the sarcin domain of 23S rRNA of Escherichia coli ribosomes was determined by a combinatory approach of oligo DNA probes and the action of alpha-sarcin. The sarcin domain is protected by a reactive complementary oligo DNA probe against the hydrolytic action of alpha-sarcin. This protective effect is dependent upon the length and the complementary sequence of oligo DNA probes that react to ribosomes. Under UV irradiation and using of the primer extension, nucleotides that contacted by reactive oligo DNA probes were determined. Nucleotides at the 3' side of the domain (positions from G2659 to C2676) were targeted by oligo DNA probes that have their sequences to complement the domain, indicating that the 3' side region was exposed on the surface of ribosomes, whereas nucleotides at the 5' side of stem and extented to two bases at the loop (positions from C2646 to A2654) were not accessible to any oligo DNA probes, implying that the region could be buried in ribosomes. This study also provided evidence that the conformation of the sarcin domain is subjected to alteration if the exposed 3' side of domain is targeted by the reactive DNA probe. The importance of the topological arrangement of the sarcin domain that engages in the translocation event during translation is discussed.

The regional structure of the sarcin domain of 23S rRNA of Escherichia coli ribosomes was determined by a combinatory approach of oligo DNA probes and the action of α-sarcin. The sarcin domain is protected by a reactive complementary oligo DNA probe against the hydrolytic action of α-sarcin. This protective effect is dependent upon the length and the complementary sequence of oligo DNA probes that react to ribosomes. Under UV irradiation and using of the primer extension, nucleotides that contacted by reactive oligo DNA probes were determined. Nucleotides at the 3′ side of the domain (positions from G2659 to C2676) were targeted by oligo DNA probes that have their sequences to complement the domain, indicating that the 3′ side region was exposed on the surface of ribosomes, whereas nucleotides at the 5′ side of stem and extented to two bases at the loop (positions from C2646 to A2654) were not accessible to any oligo DNA probes, implying that the region could be buried in ribosomes. This study also provided evidence that the conformation of the sarcin domain is subjected to alteration if the exposed 3′ side of domain is targeted by the reactive DNA probe. The importance of the topological arrangement of the sarcin domain that engages in the translocation event during translation is discussed.