Interaction of antibiotics with functional sites in 16S ribosomal RNA
TL;DR: Chemical footprinting shows that several classes of antibiotics protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes, having strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA.
Abstract: Chemical footprinting shows that several classes of antibiotics (streptomycin, tetracycline, spectinomycin, edeine, hygromycin and the neomycins) protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes These findings have strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA
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TL;DR: Changing the use of tetracyclines in human and animal health as well as in food production is needed if this class of broad-spectrum antimicrobials through the present century is to continue to be used.
Abstract: Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.
3,647 citations
Cites background from "Interaction of antibiotics with fun..."
...Nevertheless, naturally occurring tetracycline-resistant propionibacteria contain a cytosine-toguanine point mutation at position 1058 in 16S rRNA (251) (see below), which does at least suggest that the neighboring bases U1052 and C1054 identified by chemical footprinting (180) may have functional significance for the binding of tetracyclines to the 30S subunit....
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...Several studies have indicated a single, high-affinity binding site for tetracyclines in the ribosomal 30S subunit, with indications through photoaffinity labeling and chemical footprinting studies that protein S7 and 16S rRNA bases G693, A892, U1052, C1054, G1300, and G1338 contribute to the binding pocket (44, 180, 196, 263)....
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TL;DR: The crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution is described, suggesting coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.
Abstract: We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.
1,933 citations
TL;DR: Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity.
Abstract: Nucleic acid molecules play crucial roles in diverse biological processes including the storage, transport, processing, and expression of the genetic information. Nucleic acid aptamers are selected in vitro from libraries containing random sequences of up to a few hundred nucleotides. Selection is based on the ability to bind ligand molecules with high affinity and specificity. Three-dimensional structures have been determined at high resolution for a number of aptamers in complex with their cognate ligands. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity. These basic principles of discriminatory molecular interactions in aptamer complexes parallel recognition events central to many cellular processes involving nucleic acids.
1,547 citations
TL;DR: The functional implications of the high-resolution 30S crystal structure are described, and details of the interactions between the 30S subunit and its tRNA and mRNA ligands are inferred, which lead to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.
Abstract: The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.
1,508 citations
TL;DR: Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.
Abstract: We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 108-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.
1,329 citations
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TL;DR: The primary structure of rRNA Species-Nomenclature and the secondary structure of Secondary-Structure are investigated in more detail in this chapter.
Abstract: PERSPECTIVES AND SUMMARy............................................................................................................. 119 rRNA Species-Nomenclature .. ... ... ... ... .... .. ............................ ... ... ..... .. ... .... ... .. ... .... .... . 120 PRIMARY STRUCTURE............................................................................................................................ 121 SECONDARY STRUCTURE....................................................................................................................... 124 General Approaches ....................... ... .. .. ... .. ... .. . . . .. ........................ .... ... .. .... .. .... ... . .. ... .. 124 Description of Secondary-Structure M ode/s......... ... ....... ... ........... ... .. . ....... ..................... .. 129 Phylogenetic Comparison ............ . .. . .... ... .. ... .. . .... ... .. .................... ... ... ... ... .. . ... .... ..... .. .. 136 Experimental Tests.... . . .. .. ....... ........ ... .... .. . .. ... ................ ... .. ... ........ .... .. .... ............... .. . ... 136
765 citations
TL;DR: Four different base-specific chemical reactions generate a means of directly sequencing RNA terminally labeled with 32P, which yields clean cleavage patterns for each purine and pyrimidine and allows a determination of the entire RNA sequence out to 100-200 bases from the labeled terminus.
Abstract: Four different base-specific chemical reactions generate a means of directly sequencing RNA terminally labeled with 32P. After a partial, specific modification of each kind of RNA base, an amine-catalyzed strand scission generates labeled fragments whose lengths determine the position of each nucleotide in the sequence. Dimethyl sulfate modifies guanosine. Diethyl pyrocarbonate attacks primarily adenosine. Hydrazine attacks uridine and cytidine, but salt suppresses the reaction with uridine. In all cases, aniline induces a subsequent strand scission. The electrophoretic fractionation of the labeled fragments on a polyacrylamide gel, followed by autoradiography, determines the RNA sequence. RNA labeled at the 3' end yields clean cleavage patterns for each purine and pyrimidine and allows a determination of the entire RNA sequence out to 100-200 bases from the labeled terminus.
744 citations
TL;DR: In this paper, the authors examined the range of the variation of secondary structure among the 16-S-like rRNAs and provided a basis for an accurate alignment of the corresponding regions of different primary structures.
Abstract: Publisher Summary This chapter examines the range of the variation of secondary structure among the 16-S-like rRNAs. This brings into a larger structural context a recent detailed analysis of the individual helical elements and provides a basis for an accurate alignment of the corresponding regions of different primary structures. Computer-assisted comparative is used in the analysis of aligned sequences to describe the pattern of phylogenetic conservation for each nucleotide position in 16-S rRNA. A search for matching patterns among unpaired positions in the RNA chain then produces a list of candidates for potential base–base tertiary interactions. The completion of nucleotide sequences for 34 16-S-like rRNAs includes 4 eubacteria, 4 chloroplasts, 12 mitochondria, 4 archaebacteria, and 10 eukaryotes. Secondary structure models for these molecules have been developed in the course of refinement of the E. coli model, and have been used to arrive at improved sequence alignments for the 16-S-like rRNAs. Schematic drawings of (1) eubacterial, (2) archaebacterial, (3) eukaryotic cytoplasmic, (4) plant mitochondrial, (5) fungal mitochondrial, and (6) mammalian mitochondrial structures are shown in the chapter.
693 citations
TL;DR: Etude des differents elements helicoidaux : Structures d'ordre superieur a la structure secondaire.
629 citations