Protection of specific sites in 16 S RNA from chemical modification by association of 30 S and 50 S ribosomes.

The effect of 30S subunit attachment on the accessibility of specific sites in 5 S and 23 S RNA in 50 S ribosomal subunits was studied by means of the guanine-specific reagent kethoxal. Oligonucleotides surrounding the sites of kethoxal substitution were resolved and quantitated by diagonal electrophoresis. In contrast to the extensive protection of sites in 16 S RNA in 70 S ribosomes (Chapman & Noller, 1977), only two strongly (approx. 90%) protected sites were detected in 23 S RNA. The nucleotide sequences at these sites are in which the indicated kethoxal-reactive guanines (with K above them) are strongly protected by association of 30 S and 50 S subunits. The latter sequence has the potential to base-pair with nucleotides 816 to 821 of the 16 S RNA, a site which has been shown to be protected from kethoxal by 50 S subunits and essential for subunit association. Six additional sites in 23 S RNA are partially (30 to 50%) protected by 30 S subunits. One of these sequences, is complementary to nucleotides 787 to 792 of 16 S RNA. a site which is also 50 S-protected and essential for association. Of the two kethoxal-reactive 5 S RNA sites in 50 S subunits, G13 is partially protected in 70 S ribosomes. while G41 remains unaffected by subunit association. The relatively small number of kethoxal-reactive sites in 23 S RNA that is strongly protected in 70 S ribosomes suggests that subunit association may involve contacts between single-stranded sites in 16 S RNA and 50 S subunit proteins or non-Watson-Crick interactions with 23 S RNA. in addition to the two suggested base-paired contacts.

[1]  H. Noller,et al.  Nucleotide sequences of accessible regions of 23S RNA in 50S ribosomal subunits. , 1978, Biochemistry.

[2]  G G Brownlee,et al.  The sequence of 5 s ribosomal ribonucleic acid. , 1968, Journal of molecular biology.

[3]  P. Stiegler,et al.  The determination of the primary structure of the 16S ribosomal RNA of Escherichia coli. 2. Nucleotide sequences of products from partial enzymatic hydrolysis. , 1972, Biochimie.

[4]  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.

[5]  A. Zamir,et al.  Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function. , 1975, Journal of Molecular Biology.

[6]  C. Woese,et al.  Conservation of primary structure in 16S ribosomal RNA , 1975, Nature.

[7]  J. Steitz.  Polypeptide Chain Initiation: Nucleotide Sequences of the Three Ribosomal Binding Sites in Bacteriophage R17 RNA , 1969, Nature.

[8]  J. Davies,et al.  Change in methylation of 16S ribosomal RNA associated with mutation to kasugamycin resistance in Escherichia coli. , 1971, Nature: New biology.

[9]  C. Kurland,et al.  30S Ribosomal proteins associated with the 3′‐terminus of 16S RNA , 1975, FEBS letters.

[10]  C. Levinthal,et al.  A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. , 1960, Biochimica et biophysica acta.

[11]  H. Noller,et al.  Letters to the editor: Accessibility of 5 S RNA in 50 S ribosomal subunits. , 1974, Journal of molecular biology.

[12]  S. Nishimura,et al.  Isolation and characterization of 2-methyladenosine from Escherichia coli tRNAGlu2, tRNAAsp1, tRNAHis1 and tRNAArg , 1972 .

[13]  H. Noller.  Topography of 16S RNA in 30S ribosomal subunits. Nucleotide sequences and location of sites of reaction with kethoxal. , 1974, Biochemistry.

[14]  P. Stiegler,et al.  Primary sequence of the 16S ribosomal RNA of Escherichia coli. , 1975, Nucleic acids research.

[15]  P. Fellner.  Nucleotide sequences from specific areas of the 16S and 23S ribosomal RNAs of E. coli. , 1969, European journal of biochemistry.

[16]  B. D. Davis,et al.  Actions of aurintricarboxylate, kasugamycin, and pactamycin on Escherichia coli polysomes. , 1973, Biochemistry.

[17]  M. Santer,et al.  Area of 16S ribonucleic acid at or near the interface between 30S and 50S ribosomes of Escherichia coli , 1977, Journal of bacteriology.

[18]  R. Kenner.  A protein-nucleic acid crosslink in 30S ribosomes. , 1973, Biochemical and biophysical research communications.

[19]  G. Craven,et al.  Identification of neighbor relationships among proteins in the 30 S ribosome: intermolecular cross-linkage of three proteins induced by tetranitromethane. , 1973, Journal of molecular biology.

[20]  H. Noller,et al.  Altered topography of 16S RNA in the inactive form of Escherichia coli 30S ribosomal subunits. , 1978, Biochemistry.

[21]  C. Kurland,et al.  New aspects of the IF3‐ribosome interaction , 1976, FEBS letters.

[22]  B. Hapke,et al.  Structural dynamics of bacterial ribosomes. I. Characterization of vacant couples and their relation to complexed ribosomes. , 1973, Journal of molecular biology.

[23]  H. W. Schaup,et al.  Localization of a Binding Site for Ribosomal Protein S8 Within the 16S Ribosomal Ribonucleic Acid of Escherichia coli , 1973, Journal of bacteriology.

[24]  R. Garrett,et al.  Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli. , 1975, Nucleic acids research.

[25]  I. Holland,et al.  Colicin E3-directed changes in ribosome function and polyribosome metabolism in Escherichia coli K12. , 1970, Journal of molecular biology.

[26]  F. Chang,et al.  The specific cross-linking of two proteins from the Escherichia coli 30 s ribosomal subunit. , 1972, Journal of molecular biology.

[27]  J. Ebel,et al.  The determination of the primary structure of the 16S ribosomal RNA of Escherichia coli. 1. Nucleotide sequence analysis of T 1 and pancreatic ribonuclease digestion products. , 1972, Biochimie.

[28]  M. Litt.  Structural studies on transfer ribonucleic acid. I. Labeling of exposed guanine sites in yeast phenylalanine transfer ribonucleic acid with kethoxal. , 1969, Biochemistry.

[29]  M. Nomura,et al.  Assembly Mapping of 30S Ribosomal Proteins from E. coli , 1970, Nature.

[30]  R. Kohler,,et al.  Polysomes Extracted from Escherichia coli by Freeze-Thaw-Lysozyme Lysis , 1966, Science.

[31]  J. Dunn,et al.  The reaction of 5S RNA in 70S ribosomes with kethoxal , 1975, FEBS letters.

[32]  The binding site of protein L1 on 23-S ribosomal RNA from Escherichia coli. 3. Nucleotide sequence. , 1976 .

[33]  R. Garrett,et al.  Ribosomal proteins. XXVI. The number of specific protein binding sites on 16 s and 23 s RNA of Escherichia coli. , 1971, Journal of molecular biology.