title: Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?
abstract: Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the rrnB operon of E. coli was subjected to primer-directed mutagenesis. To produce the deletion it was necessary to use simultaneously the mutagenic dodecamer dCGGCGCACGGCG and the universal M13 primer dCCCAGTCACGACGTT, and to employ forced annealing conditions. The mutated gene was expressed in an overproducing plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired adenosine and the adjacent guanosine-67 of the RNA and glutamine-19 of the protein L18.
authors.forename: J
authors.initials: J
authors.lastname: Christiansen
authors.forename: S R
authors.initials: SR
authors.lastname: Douthwaite
authors.forename: A
authors.initials: A
authors.lastname: Christensen
authors.forename: R A
authors.initials: RA
authors.lastname: Garrett
mesh_terms: D000241:Adenosine; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006860:Hydrogen Bonding; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012335:RNA, Ribosomal / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D013698:Templates, Genetic
chemical_list: D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C029609:ribosomal protein L18; D004262:DNA Restriction Enzymes; D000241:Adenosine