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J. Biol. Chem., Vol. 281, Issue 43, 32303-32309, October 27, 2006

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The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation^*

Taeko Komoda, Neuza S. Sato¹, Steven S. Phelps^¶, Naoki Namba, Simpson Joseph^¶, and Tsutomu Suzuki²

From the Department of Chemistry and Biotechnology, Graduate School of Engineering, and Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan and the ^¶Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0314

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

Received for publication, July 25, 2006 , and in revised form, August 25, 2006.

^* This work was supported by National Institutes of Health Grant GM65265 and National Science Foundation Grant 0315780 (both to S. J.), grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan (to T. S.), and by the Human Frontier Science Program (RGY23/2003) (to T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Institute Adolfo Lutz, Ave. Dr. Arnaldo 355 10th floor, 01246-902 Sao Paulo, SP, Brazil.

² To whom correspondence should be addressed. Tel.: 81-3-5841-8752; Fax: 81-3-3816-0106; E-mail: ts{at}chembio.t.u-tokyo.ac.jp.

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