title: The phenotype of mutations of the base-pair C2658.G2663 that closes the tetraloop in the sarcin/ricin domain of Escherichia coli 23 S ribosomal RNA.
abstract: The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA is a part of the site for the association of elongation factors with ribosomes and for that reason is critical for the binding of aminoacyl-tRNA and for translocation during the reiterative elongation reactions of protein synthesis. The SRD has a GAGA tetraloop that is shut off by a Watson-Crick C2658 x G2663 pair. The contribution of this pair to the function of the ribosome has been evaluated by constructing mutations in the nucleotides and determining their phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a G2663C transversion mutation that disrupts the Watson-Crick pair was lethal. Double transversion mutations, C2658G x G2663C and C2658A x G2663U, that reverse the polarity of the pyrimidine and the purine but restore the potential to form a canonical pair, were also lethal. Induction of transcription of 23 S rRNA with the same mutations, but encoded in a plasmid with a lambdaP(L) promoter and expressed at a lower level, retarded growth. The sedimentation profiles of ribosomes with transversion mutations in C2658 and/or G2663 are altered; the ratio of 50 S subunits to 30 S particles is changed and polysomes are reduced. Ribosomes with a G2663C, a C2658G x G2663C, or a C2658A x G2663U mutation in 23 S rRNA were not active in protein synthesis, indeed, they appeared to inhibit the activity of ribosomes with wild-type 23 S rRNA. Transversion mutations in the analogs of C2658 and G2663 decreased binding of EF-G to SRD oligoribonucleotides; the same mutations in 23 S rRNA decreased binding of the factor to intact ribosomes. The most severe phenotype, in growth, in protein synthesis, and in the binding of EF-G, was associated with a C2658G x G2663C mutation; it is surprising that this was more severe than an analogous C2658A x G2663U mutation. A double transition mutation, C2658U x G2663A, which is not known to have occurred in nature, had no effect on the growth of cells or on the function of ribosomes. The lethal phenotype of transversion mutations in C2658 and G2663 appears to derive from a loss of the capacity of ribosomes to bind EF-G and by indirection the EF-Tu ternary complex.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Y L
authors.initials: YL
authors.lastname: Chan
authors.forename: A S
authors.initials: AS
authors.lastname: Sitikov
authors.forename: I G
authors.initials: IG
authors.lastname: Wool
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism; D020029:Base Pairing / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005656:Fungal Proteins; D005804:Genes, Lethal / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020653:Peptide Elongation Factor G / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D012276:Ricin / Q000378:metabolism*; D013816:Thermodynamics; D012407:rRNA Operon / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D009843:Oligoribonucleotides; D020653:Peptide Elongation Factor G; D010455:Peptides; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; C014801:alpha-sarcin; C012001:polyphenylalanine; D006160:Guanosine Triphosphate; D012276:Ricin; D004722:Endoribonucleases