Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors.

Alpha-sarcin cleaves one phosphodiester bond of 23S rRNA within 70S ribosomes or 50S subunits derived from E. coli. The resulting fragment was isolated and sequenced. The cleavage site was identified as being after G2661 and is located within a universally conserved dodecamer. Cleavage after G2661 specifically blocked the binding of both elongation factors, i.e. that of the ternary complex Phe-tRNA*EF-Tu*GMPPNP and of EF-G*GMPPNP, whereas all elongation-factor independent functions of the ribosome, such as association of the ribosomal subunits, tRNA binding to A and P sites, the accuracy of tRNA selection at both sites, the peptidyl transferase activity, and the EF-G independent, spontaneous translocation, were not affected at all. Control experiments with wheat germ ribosomes yielded an equivalent inhibition pattern. The data suggest that the universally conserved dodecamer containing the cleavage site G2661 is located at the presumably overlapping region of the binding sites of both elongation factors.

[1]  G. Kramer,et al.  Structure, Function, and Genetics of Ribosomes , 1986, Springer Series in Molecular Biology.

[2]  I. Wool,et al.  The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates. , 1983, The Journal of biological chemistry.

[3]  D. Richter.  Competition between the elongation factors 1 and 2, and phenylalanyl transfer ribonucleic acid for the ribosomal binding sites in a polypeptide-synthesizing system from brain. , 1973, The Journal of biological chemistry.

[4]  H. Rheinberger,et al.  The ribosomal elongation cycle: tRNA binding, translocation and tRNA release. , 1983, European journal of biochemistry.

[5]  J. Davies,et al.  Specific cleavage of ribosomal RNA caused by alpha sarcin. , 1977, Nucleic acids research.

[6]  K. Nierhaus,et al.  Isolation of the protein synthesis elongation factors EF-Tu, EF-Ts, and EF-G from Escherichia coli. , 1979, Methods in enzymology.

[7]  K. Nierhaus,et al.  Spontaneous, elongation factor G independent translocation of Escherichia coli ribosomes. , 1983, The Journal of biological chemistry.

[8]  F. Dohme,et al.  [37] Total reconstitution of 50 S subunits from Escherichia coli ribosomes , 1979 .

[9]  K. Nierhaus,et al.  Minimal set of ribosomal components for reconstitution of the peptidyltransferase activity. , 1982, The EMBO journal.

[10]  O. Uhlenbeck,et al.  3′-Terminal labelling of RNA with T4 RNA ligase , 1978, Nature.

[11]  U. Geigenmüller,et al.  Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined. , 1986, European journal of biochemistry.

[12]  C. Woese,et al.  Complete nucleotide sequence of a 23S ribosomal RNA gene from Bacillus stearothermophilus. , 1984, DNA.

[13]  I. Wool,et al.  The primary structure of the cytotoxin alpha-sarcin. , 1983, The Journal of biological chemistry.

[14]  I. Wool,et al.  The site of action of alpha-sarcin on eukaryotic ribosomes. The sequence at the alpha-sarcin cleavage site in 28 S ribosomal ribonucleic acid. , 1982, The Journal of biological chemistry.

[15]  H. Ceri,et al.  Association of a ribonuclease with the 50-S ribosomal subunit of Escherichia coli MRE 600. , 1973, Biochimica et biophysica acta.

[16]  K. Edwards,et al.  The rRNA operon from Zea mays chloroplasts: nucleotide sequence of 23S rDNA and its homology with E.coli 23S rDNA. , 1981, Nucleic acids research.

[17]  W. Gilbert,et al.  Mapping adenines, guanines, and pyrimidines in RNA. , 1977, Nucleic acids research.

[18]  R. Brimacombe,et al.  Nucleotide sequences of Escherichia coli 16-S RNA associated with ribosomal proteins S7, S9, S10, S14 and S19. , 1975, European journal of biochemistry.

[19]  Richard Brimacombe,et al.  Application of a rapid gel method to the sequencing of fragments of 16S ribosomal RNA from Escherichia coli , 1978, Nucleic Acids Res..

[20]  H. Rheinberger,et al.  Allosteric interactions between the ribosomal transfer RNA-binding sites A and E. , 1986, The Journal of biological chemistry.

[21]  E. Cundliffe,et al.  The mode of action of alpha sarcin and a novel assay of the puromycin reaction. , 1978, The Biochemical journal.

[22]  M. Sugiura,et al.  The complete nucleotide sequence of a 23-S rRNA gene from tobacco chloroplasts. , 1982, European journal of biochemistry.

[23]  K. Nierhaus,et al.  A method for the 14C-labeling of proteins from the large ribosomal subunit, without loss of biological activity. , 1979, Methods in Enzymology.

[24]  I. Wool.  The mechanism of action of the cytotoxic nuclease α-sarcin and its use to analyse ribosome structure , 1984 .

[25]  R. Brimacombe,et al.  Refined secondary structure models for the 16S and 23S ribosomal RNA of Escherichia coli. , 1983, Nucleic acids research.

[26]  R. Brimacombe,et al.  Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli. , 1977, European Journal of Biochemistry.

[27]  C. Fernández-Puentes,et al.  Effects of some proteins that inactivate the eukaryotic ribosome , 1977, FEBS letters.

[28]  R. Dale,et al.  Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions. , 1984, Plasmid.

[29]  A. Rich,et al.  Protein synthesis with ribonuclease digested ribosomes. , 1972, Biochimica et biophysica acta.

[30]  R. Amils,et al.  Sensitivity of thermoacidophilic archaebacteria to α‐sarcin , 1984 .

[31]  V. Nosikov,et al.  Primary and secondary structure of rat 28 S ribosomal RNA. , 1984, Nucleic acids research.

[32]  K. Skryabin,et al.  The structure of the yeast ribosomal RNA genes. 4. Complete sequence of the 25 S rRNA gene from Saccharomyces cerevisae. , 1981, Nucleic acids research.

[33]  R. A. Etten,et al.  Precise localization and nucleotide sequence of the two mouse mitochondrial rRNA genes and three immediately adjacent novel tRNA genes , 1980, Cell.