title: Mapping ribosomal protein S20-16 S rRNA interactions by mutagenesis.
abstract: Specific ribonucleotides within the 5' domain of Escherichia coli 16 S rRNA were altered by deletion and/or substitution by site-directed mutagenesis of cloned DNA to assess the importance of these particular residues in defining the binding site for ribosomal protein S20. Gel filtration and sucrose gradient centrifugation were employed to measure the binding of ribosomal protein S20 to synthetic transcripts spanning the 5' domain of 16 S rRNA containing various alterations. In several cases, the apparent association constants for these interactions were also determined. Three essential results were obtained. First, RNA transcripts containing residues 1-402 are sufficient for high affinity S20 binding. Second, bulges at residues 250-251 and 278-280 in a stem-loop structure extending from residues 240 to 286 are critical for S20 binding, arguing that the "260 stem" directly interacts with S20 at these sites. Third, mutations in a hairpin structure spanning residues 316-337 demonstrate a strong requirement for both the specific A321*G332 bulge and for unpaired residues in the loop itself for proper S20 binding. Our results document the importance of unpaired residues in maintaining the interaction between S20 and 16 S rRNA.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Canada.
authors.forename: R S
authors.initials: RS
authors.lastname: Cormack
authors.forename: G A
authors.initials: GA
authors.lastname: Mackie
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C029611:ribosomal protein S20