Further evidence that the ribosomal 30S proteins S3, S5, S9, S11, S12, and S18 possess specific 16S RNA binding sites

Summary E. coli ribosomal 16S RNA preparted by an acetic acid-urea extraction technique individually binds, in addition to the seven established proteins, 6 new 30S ribosomal proteins (S3, S5, S9, S12, S18 and S11) (Hochkeppel et al., 1976). In this communication we demonstrate the site specificity of these proteins. Binding curves of the individual proteins with acetic acid-urea 16S RNA show that the binding of all six proteins to the RNA reaches a plateau at 0.3–0.97 copies per 16S RNA molecule. No significant binding of these proteins to classical phenol extracted 16S RNA is observed, with the exception of S13 which binds 0.2 copies of protein per molecule of 16S RNA. Specificity of binding of these proteins is also demonstrated in chase experiments. The site specificity of individual [³H]-labeled 30S proteins bound to 16S RNA is tested by the addition of non-radioactive 30S total protein to the reaction mixture.     

Molecular interactions of ribosomal components

Summary The site-specific complex formed between 16S RNA and the 30S ribosomal protein S4 from Escherichia coli has been degraded with pancreatic ribonuclease. We have recovered the nuclease-resistant RNA from this complex; we call it S4aR. S4aR will bind to S4, but it will not bind to the other 30S proteins that can form site-specific complexes with 16S RNA. The data presented here as well as elsewhere (Schaup et al., 1971b) show that S4aR has a mass of about 150000 daltons and that it is made up of several separate RNA fragments, each of which enters the complex with S4. We conclude that S4 interacts with several separate binding sites on the RNA and that these probably contain a great deal of double stranded structure.     

Methylation status of 13S ribosomal RNA from hamster mitochondria: the presence of a novel riboside, N4-methylcytidine.

The ribosomal RNA ("13S" RNA) of the small ribosomal subunit of hamster cell mitochondria has been found to have a distinctive pattern of methylated residues. Each molecule contained, on the average, approximately one residue of m4Cp, m5Cp and m5Up, and two residues of m62Ap. The natural occurrence of m4Cp has not previously been reported; we propose that this nucleotide is homologous to its ribose-methylated congener, m4Cmp, which is characteristic of bacterial 16S ribosomal RNA. We detected neither m4Cp nor m4Cmp in the hamster cell cytoplasmic ribosomal RNA. This is the first documentation of a modified residue present in mitochondrial RNA but absent from the cytoplasmic RNA of the same cells.     

Molecular cloning of the ribosomal RNA genes of the photosynthetic bacterium Rhodopseudomonas capsulata

Summary Chromosomal segments of Rhodopseudomonas capsulata carrying the ribosomal operons and cloned with the cosmid vector pHC79 have been identified by cross hybridization with ³²P-ATP labeled rRNAs. At least seven rRNA operons are present in the R. capsulata chromosome. By R-loop analyses of DNA-RNA hybrids, two distinct loop structures of sizes 1.50 kb and 2.52 kb corresponding to the 16S and 23S RNA molecules, respectively, were detected. Intact 23S RNA molecules can be isolated from R. capsulata ribosomes by sucrose density centrifugation. However, fragmentation of the 23S RNA molecule into a 16S-like molecule was observed during gel electrophoresis. Restriction mapping and hybridization of a 9 kb PstI fragment that contained one copy of the rRNA operon showed the following sequence of the RNA genes in R. capsulata 16S, 23S, and 5S. A spacer region of 0.91 kb was found between the 16S and the 23S RNA genes.     

Mechanism of Kasugamycin Resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Strains of Escherichia coli are resistant to the antibiotic kasugamycin due to the partial non-methylation of 16S ribosomal RNA. An RNA methylase activity, absent from resistant strains, is shown here to methylate in vitro the 16S RNA of resistant as well as sensitive strains.     

Additional Nucleotide Sequences in Precursor 16S Ribosomal RNA from <Emphasis Type="Italic">Escherichia coli</Emphasis>

MATURE 5S, 16S and 23S ribosomal RNA species present in E. coli ribosomes are the end products of complex biosyn-thetic pathways. They are formed by reduction in length, and methylation of longer RNA chains transcribed on the ribosomal RNA cistrons of E. coli DNA. While these modifications take place the ribosome structure is formed by progressive addition of ribosomal proteins and conformational changes in the resulting ribonucleoprotein precursor particles¹.     

Characterization of the ribosomal ribonucleic acids of blue-green algae

Summary The ribosomal RNA components of 12 species of blue-green algae have been characterized. The 23S RNA of most species is labile and discrete cleavage products were detected by polyacrylamide gel electrophoresis. In contrast, the 23S and 16S RNA's of three species, Anacystis nidulans, Nostoc sp. and Oscillatoria tenuis were essentially undegraded (apart from a hidden break in some of the 23S RNA molecules) and these are the most suitable species for further study. The undegraded 23S and 16S RNA's have similar molecular weights (1.07×10⁶ and 0.53–0.54×10⁶ respectively) to the corresponding molecules from bacteria and eukaryote chloroplasts. The nucleotide base compositions of separated, intact, 23S and 16S RNA's from blue-green algae are also of the prokaryotic type. For instance, the (G+C) content of each RNA is approximately 52 moles % and the (G-C)+(A-U) values are high (16–24 moles %). Blue-green algae, like other organisms, contain a 5S ribosomal RNA. Its electrophoretic mobility in polyacrylamide gels and its behaviour on methylated-albumen-kieselguhr-columns relative to E. coli, plant cytoplasmic and plant chloroplast 5S RNA's, are described.     

30S ribosomal proteins cross-linked to 16S RNA by periodate oxidation followed by borohydride reduction

Gel electrophoretic techniques have been used to reexamine the RNA-protein cross-linking reaction induced by periodate oxidation and borohydride reduction of 30S ribosomal subunits. The results show that a number of 30S ribosomal proteins become attached to intact 16S RNA by this method, in addition to those already published. It follows that this cross-linking technique as it stands is of little value as a topographical probe of the environment of the 3-terminus of the 16S RNA.     

Comparison of ribosomal RNA-binding protein "L2" isolated from different bacterial species

Summary A ribosomal protein (L2) which binds to 23S rRNA was isolated from 70S ribosomes of several Bacillacease. It was shown by two-dimensional gel electrophoresis, molecular weight determination, amino acid analysis and immunological methods that this protein is homologous to the E. coli ribosomal protein L2 which also binds to 23S rRNA. In all Bacillaceae this protein L2 remains bound to 23S rRNA after extraction of ribosomal proteins with 4 M urea and 2 M LiCl, in contrast to E. coli. Immunological experiments demonstrated that the protein L2 of B. stearothermophilus undergoes a conformational change when it binds to 23S RNA.Paper No. 69 on Ribosomal proteins. Preceding paper is by Geisser et al., Molec. gen. Genet. 127, 129–145 (173)     

Isolation and characterization of a 5S RNA-protein complex from human placental ribosomes

Large ribosomal subunits treated with EDTA change their sedimentation rate in sucrose gradients and lose the 5S RNA molecule, which is released in a ribonucleoprotein particle sedimenting at about 7S. The proteins bound in this complex were identified by two-dimensional (2-D) polyacrylamide gel electrophoresis as ribosomal proteins L3 and L4, both having a molecular weight of about 37000.     

The arrangement of nucleotides in the coding regions of natural templates

Summary The relation of the nucleotide sequences in the coding regions of natural templates and of the short nucleotide sequence in 3 terminus of 16 S ribosomal RNA was found to differ from random pattern. The observation is interpreted in terms of both the ribosomal interactions and the molecular evolution.     

Characterization of cloned rat ribosomal DNA fragments

Summary Two Charon 4A lambda bacteriophage clones were characterized which contain all and part of the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA region were in the same places in mouse and rat DNA but the restriction sites in the 5-spacer regions were different.     

Protection of specific sites in 23 S and 5 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.     

Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin.

Summary Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.     

Mutant Ribosomal Protein with Defective RNA Binding Site

THE 30S ribosomal subunits of Escherichia coli contain twenty-one different proteins^1–4, which together with 16S RNA can reassemble in vitro to form functional 30S particles⁵. Five proteins can individually bind to specific sites on the 16S RNA^6–8 and these are S4, S7, S8, S15 and S20 (in the nomenclature recently adopted by several laboratories to report results with the E. coli system⁹). We report here the first identification of a mutation that affects a ribosomal protein-nucleic acid interaction.     

Organisation of the ribosomal RNA genes in Streptomyces coelicolor A3(2)

Summary Using Southern hybridisation of radiolabelled purified ribosomal RNAs to genomic DNA the ribosomal RNA genes of Streptomyces coelicolor A3(2) were shown to be present in six gene sets. Each gene set contains at least one copy of the 5 S, 16 S and 23 S sequences and in at least two cases these are arranged in the order 16 S-23S-5S. Three gene sets, rrnB, rrnD and rrnF, were isolated by screening a library of S. coelicolor A3(2) DNA. The restriction map of one of these, rrnD, was determined and the nucleotide sequences corresponding to the three rRNAs were localised by Southern hybridisation. The gene order in rrnD is 16S-23S-5S.