Characterization of in vitro and in vivo mutations in non-conserved nucleotides in the ribosomal RNA recognition domain for the ribotoxins ricin and sarcin and the translation elongation factors.
The sarcin/ricin domain in 23 S/28 S rRNA is crucial for ribosome function, since it constitutes at least part of the binding site for the elongation factors and hence is essential for binding aminoacyl-tRNA and for translocation. The domain is also the site of action of ricin and sarcin and analysis of the effect of mutations in the RNA on recognition by the cytotoxins has helped to define the structure and to understand the function of the region. We have constructed deletions, separately, of pairs of non-conserved, juxtaposed but non-hydrogen-bonded nucleotides that correspond to C4317 and C4331, and to U4316 and C4332, in an oligoribonucleotide that mimics the sarcin/ricin domain in rat 28 S rRNA. The deletions had no effect on the depurination of A4324 by ricin nor on the cleavage of the phosphodiester bond on the 3' side of G4325 by sarcin. However, simultaneous deletion of the four nucleotides decreased cleavage by sarcin but did not affect depurination by ricin. Removal of the non-canonical A4318.A4330 pair abolished recognition by both toxins. Deletion from oligoribonucleotides, that reproduce the sarcin/ricin domain of Escherichia coli 23 S rRNA, of U2653 and C2667 (equivalent to U4316, C4317 and C4331, C4332 in 28 S rRNA), or substitution of guanosine for U2653 (designed to form a Watson-Crick G2653.C2667 pair), reduced cleavage by sarcin whereas depurination by ricin was slightly increased. An increase in the stability of the mutant oligoribonucleotides may be the basis of the impairment in sarcin action. The tm for the wild-type RNA is 60 degreesC; for the double-deletion mutant U2653Delta/C2667Delta it is 65 degreesC; and for the U2653G transversion it is 69 degreesC. Expression of a mutant 23 S rRNA gene lacking U2653 and C2667 is lethal and a U2653G transversion mutation impairs growth. The mutant ribosomes are less active in protein synthesis than the wild-type and ribosomes with the U2653G mutation are resistant to sarcin. The binding of EF-G to oligoribonucleotides with a U2653/C2667 double deletion is reduced and an effect on the affinity of the factor for the sarcin/ricin domain may account in part for the decrease in ribosome efficiency. The results stress the potential importance in rRNA structure and function of non-conserved nucleotides, and suggest that the sarcin/ricin domain in ribosomes requires a region of structural flexibility for optimal efficiency.
Macbeth
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MR
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, 60637, USA.
Wool
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IG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009711:Nucleotides; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D014176:Protein Biosynthesis; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012339:RNA, Ribosomal, 28S / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012276:Ricin / Q000378:metabolism*; D012407:rRNA Operon
D005656:Fungal Proteins; D009711:Nucleotides; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012339:RNA, Ribosomal, 28S; C014801:alpha-sarcin; D012276:Ricin; D004722:Endoribonucleases