The Phenotype of Mutations of G2655 in the Sarcin/Ricin Domain of 23 S Ribosomal RNA

Abstract

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA forms a part of the site for the association of the elongation factors with the ribosome and hence is critical for the binding of aminoacyl-tRNA and for translocation. The domain is also the site of action of the eponymous toxins which catalyze covalent modification of single nucleotides that inactivate the ribosome. The conformation of the conserved guanosine at position 2655 is an especially prominent feature of the structure of the SRD: the nucleotide is bulged out of a helix and forms a base-triple with A2665 and U2656. G2655 in 23 S rRNA is protected from chemical modification when the elongation factors, EF-Tu and EF-G, are bound to ribosomes and the analog of G2655 in oligoribonucleotides is critical for recognition by the toxin sarcin and by EF-G. The contribution of G2655 to the function of the ribosome has been evaluated by constructing mutations in the nucleotide and determining the phenotype. Constitutive expression of a plasmid-encoded rrn B operon with a deletion of, or transversions in, G2655 is lethal to E. coli cells, whereas a defect in the growth of cells with a G2655A transition is observed only in competition with wild-type cells. The sedimentation profiles of ribosomes with mutations in G2655 are altered; most markedly by deletion or transversion of the nucleotide, less severely by transition to adenosine. Mutations of G2655 confer resistance to sarcin on ribosomes. Ribosomes with G2655Δ, G2655C, or G2655U mutations in 23 S rRNA are not active in protein synthesis, whereas those with the G2655A transition mutation suffer decreased activity.