Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA
| Autor: | Nils G. Walter, Miguel J. B. Pereira, Meredith Newby Lambert, John A. H. Hoerter |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Ribosomal Proteins
Guanine Ultraviolet Rays Biology Slippery sequence Nucleic Acid Denaturation Biochemistry Ribosome RNA Ribosomal 16S Fluorescence Resonance Energy Transfer 30S Protein secondary structure Nuclear Magnetic Resonance Biomolecular Oligonucleotide Escherichia coli Proteins RNA Ribosomal RNA Tetracycline Crystallography Kinetics RNA Bacterial Förster resonance energy transfer Spectrometry Fluorescence Biophysics Nucleic Acid Conformation Thermodynamics Ribosomes |
| Zdroj: | Biochemistry. 43(46) |
| ISSN: | 0006-2960 |
| Popis: | The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation. Recent crystallographic and genetic evidence has called this hypothesis into question. To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E. coli H27 for in vitro physicochemical studies. One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl). UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link. The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12. FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit. Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations. This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome. |
| Databáze: | OpenAIRE |
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