In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA.
Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.
Hirao
Ichiro
I
Yokoyama CytoLogic Project, ERATO, JST, c/o The RIKEN Institute, Hirosawa, Wako-shi, Saitama 351-0198, Japan. ihirao@postman.riken.go.jp
Harada
Yoko
Y
Nojima
Takahiko
T
Osawa
Yutaka
Y
Masaki
Haruhiko
H
Yokoyama
Shigeyuki
S
D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002241:Carbohydrates; D003087:Colicins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D016601:RNA-Binding Proteins