In Vitro Selection of RNA Aptamers that Bind to Colicin E3 and Structurally Resemble the Decoding Site of 16S Ribosomal RNA ² Ichiro Hirao,* ,‡,§,| Yoko Harada, ‡,§ Takahiko Nojima, ‡ Yutaka Osawa, ⊥ Haruhiko Masaki, ⊥ and Shigeyuki Yokoyama* ,‡,§,@ Yokoyama CytoLogic Project, ERATO, JST, c/o The RIKEN Institute, Hirosawa, Wako-shi, Saitama 351-0198, Japan, Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, Research Center for AdVanced Science and Technology, The UniVersity of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, RIKEN Harima Institute at SPring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, and Department of Biophysics and Biochemistry, Graduate School of Science, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ReceiVed September 8, 2003; ReVised Manuscript ReceiVed January 13, 2004 ABSTRACT: 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. Colicin E3, an antibacterial protein, is encoded by plasmids within certain Escherichia coli strains and kills sensitive E. coli cells. This cytotoxic activity of colicin E3 is associated with the 11.8 kDa C-terminal ribonuclease domain (CRD) of the protein (1-4). The E. coli strains that produce colicin E3 resist the toxicity, because colicin E3 is expressed as a tight 1:1 complex with an immunity protein (5-9). The interactions between colicin E3 and the sensitive E. coli cells proceed in three steps: (i) binding of colicin E3 to a surface receptor, BtuB, on the outer bacterial membrane, (ii) trans- location of colicin E3 through the cell envelope, and (iii) cleavage at the position between adenine 1493 and guanine 1494 of 16S rRNA in ribosomes by the CRD, thereby inactivating translation. The colicin E3 cleavage site is close to the 3′ end of 16S rRNA and lies within the decoding region, which reportedly functions in the fidelity of protein synthesis by interacting with both the mRNA and tRNA located in the A site of ribosomes (10). The decoding region interacts with various molecules, such as initiation factors 1 and 3 and aminogly- coside antibiotics. The initiation factors required for the translation initiation in E. coli (11-13), and the binding of certain aminoglycoside antibiotics, such as neomycin, kana- mycin, and paromomycin, changes the structure of the decoding region, inducing codon misreading in translation (14-17). Initiation complex formation on ribosomes with the initiation factors is impaired by treatments with colicin E3 (18). On the other hand, the specific cleavage by colicin E3 is prevented by the binding of aminoglycoside antibiotics to the decoding region (19). Thus, all of these molecules interact with the decoding region, and they drastically change the ribosome function by their actions in the 16S rRNA decoding region. To illuminate the interaction between colicin E3 and 16S rRNA, the specific cleavage has been examined under several ² This work was supported by the RIKEN Structural Genomics/ Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-Aid for Scientific Research (KAKENHI 15350097) from the Ministry of Education, Culture, Sports, Science and Technology. * To whom correspondence should be addressed. I.H.: Protein Synthesis Technology Team, RIKEN Genomic Sciences Center, 1-7- 22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan (telephone, +81-45-503-9644; fax, +81-45-503-9645; e-mail, ihirao@ postman.riken.go.jp) or Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan (telephone, +81 3 5452 5442; fax, +81 3 5452 5442; e-mail, hirao@mkomi.rcast.u-tokyo.ac.jp). S.Y.: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (telephone, +81 3 5841 4413; fax, +81 3 5841 8057; e-mail, yokoyama@biochem.s.u-tokyo.ac.jp). ‡ JST. § RIKEN Genomic Sciences Center. | Research Center for Advanced Science and Technology, The University of Tokyo. ⊥ Graduate School of Agricultural and Life Sciences, The University of Tokyo. @ RIKEN Harima Institute at SPring-8 and Graduate School of Science, The University of Tokyo. 3214 Biochemistry 2004, 43, 3214-3221 10.1021/bi0356146 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/27/2004