Chemical probing of conformation in large RNA molecules. Analysis of 16 S ribosomal RNA using diethylpyrocarbonate.
Peattie & Gilbert (1980) have described an accurate and rapid gel method for assessing conformation of individual nucleotides in RNA, based on chemical modification of bases and aniline-induced strand scission. In order to extend this approach to analysis of large RNA molecules, we introduce the use of hybridization of modified RNA with DNA restriction fragments to generate RNA fragments of defined length. In principle, this permits chemical probing of conformation at any position of any RNA molecule for which a cloned DNA coding sequence is available. To illustrate the utility of this method, we use diethylpyrocarbonate to probe the reactivities of adenine residues in Escherichia coli 16 S rRNA under "native" (80 mM-potassium cacodylate (pH 7.0), 20 mM-MgCl2, 300 mM-KCl) and "quasi-secondary" (80 mM-potassium cacodylate (pH 7.0), 1 mM-EDTA) conditions. This study shows that: (1) there is generally good agreement between diethylpyrocarbonate reactivities of adenine residues in naked 16 S rRNA and a secondary structure model based on comparative sequence analysis; of 309 adenine residues probed under native conditions, only four strongly reactive residues are found in helices in the model. (2) Candidates for possible tertiary interactions are identified as adenine residues that are unpaired in the model and unreactive toward diethylpyrocarbonate under native conditions but reactive under quasi-secondary conditions. (3) An unexpectedly stable structure has been identified in the region between positions 109 and 279, where many adenine residues remain unreactive even at 90 degrees C in 80 mM-potassium cacodylate, 1 mM-EDTA. This may correspond to a structural "core" that is important for early events in ribosome assembly.
D000225:Adenine; D001345:Autoradiography; D001483:Base Sequence; D003596:Cytosine; D004047:Diethyl Pyrocarbonate; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D005561:Formates; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D006163:Ribonuclease T1
D005561:Formates; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D003596:Cytosine; D006163:Ribonuclease T1; D000225:Adenine; D004047:Diethyl Pyrocarbonate