Probing Surface Structure of Sarcin Domain on Ribosomes of <i>Escherichia coli</i> by Complementary Oligo DNAs and Ribosome-Inactivating Protein

Abstract

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.