Poster abstracts

Poster number 10 submitted by Sarah Walker

Characterization of ribosomes containing substitutions of E-site 23S rRNA nucleotide C2394

Sarah E. Walker (Department of Microbiology, The Ohio State University), Xiaofen Zhong (Department of Microbiology, The Ohio State University), Kurt Fredrick (Department of Microbiology and Ohio State Biochemistry Program, The Ohio State University)

Abstract:
Since the discovery of a third tRNA binding site, the exit (E) site, on the ribosome, two models for its function have emerged(1-3). The first model posits that the E site interacts with the tRNA moving out of the P site and thereby contributes to the mechanism of translocation(4). The second model suggests that E-site tRNA contributes to translational fidelity by stabilizing mRNA through codon-anticodon pairing until a cognate aminoacyl-tRNA is delivered to the A site, at which point the E-site tRNA rapidly dissociates(2). Recent crystal structures predict hydrogen bonding between the 3'; terminal adenosine (A76) of tRNA and C2394 of the 50S E site, and both of these nucleotides are universally conserved(5-7). In previous functional studies, a number of modifications of A76 of P-site tRNA decreased the rate of translocation, and these decreased rates correlated well with decreased E-site binding affinities(4). Here we approach the question of E-site function by mutating C2394 in a strain of E.coli that contains a single copy of the 23S gene. Ribosomes with substitutions at position 2394 are able to support growth with an ~2-fold slower growth rate, but these ribosomes are inactive in an in vitro translation assay. We also show that these substitutions decrease the rate of translocation in vitro by up to 80-fold and provide independent evidence that these mutations inhibit P/E state formation. From this we conclude that rapid translocation is dependent on C2394 of the 50S E site, consistent with a role for this site in the translocation mechanism. At the same time, the fact that these mutations confer unexpectedly mild growth phenotypes suggests that their defects are somehow compensated for in vivo.

References:
1. Rheinberger, H.J., Sternbach, H. and Nierhaus, K.H. (1981) Proc Natl Acad Sci U S A, 78, 5310-5314.
2. Rheinberger, H.J. and Nierhaus, K.H. (1983) Proc Natl Acad Sci U S A, 80, 4213-4217.
3. Robertson, J.M., Urbanke, C., Chinali, G., Wintermeyer, W. and Parmeggiani, A. (1986) J Mol Biol, 189, 653-662.
4. Lill, R., Robertson, J.M. and Wintermeyer, W. (1989) Embo J, 8, 3933-3938.
5. Korostelev, A., Trakhanov, S., Laurberg, M. and Noller, H.F. (2006) Cell.
6. Selmer, M., Dunham, C.M., Murphy Iv, F.V., Weixlbaumer, A., Petry, S., Kelley, A.C., Weir, J.R. and Ramakrishnan, V. (2006) Science.
7. Schmeing, T.M., Moore, P.B. and Steitz, T.A. (2003) RNA, 9, 1345-1352.

Keywords: ribosome, E site, translocation