Probing Escherichia coli 16S ribosomal RNA with DNA oligomers to determine functional and structural characteristics of the highly conserved 518-533 loop.

摘要

This study investigated structural and functional characteristics of the phylogenetically highly conserved 518-533 region of Escherichia coli 16S ribosomal RNA (rRNA) using complementary DNA oligomers ranging in size from hexamer to decamer. DNA binding assays demonstrated that the single-stranded, surface-exposed 518-533 loop was accessible to hybridization except for residues 525-528. These residues are also unreactive to chemical modification (Moazed et al., 1986a) suggesting their involvement in higher order molecular structure.;Ribonuclease H digestion of RNA in DNA-rRNA heteroduplexes and sequencing of the RNA to the generated cleavage site provided evidence for DNA hybridization to its complementary rRNA region.;Of the probes assayed for specific DNA binding to the 518-533 region, the DNA oligomer complementary to residues 518-526, 5;Competition assays between specific DNA oligomers versus poly(U) and a 208 ribonucleotide transcript from plasmid pNO2687 (mRNA) demonstrated effective displacement of bound DNA probes from rRNA by poly(U) and mRNA binding. The assays were unable to distinguish if oligomer displacement was dependent on direct contact between the 518-533 region and poly(U) or mRNA, or if binding of poly(U) or mRNA induced a conformational change in the probe binding site at the 518-533 region thereby displacing the oligomer. The potential for direct contact between mRNA and the 518-533 region has been proposed (Trifonov, 1987) where the 518-533 loop participates in a mRNA frame-monitoring mechanism.;Saturating concentrations of selected DNA oligomers were unable to reduce the poly(U)- or mRNA-directed incorporation of radiolabeled amino acids into trichloroacetic acid-precipitable polypeptides at reaction temperatures of 20