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Journal of Bacteriology, June 2009, p. 3445-3450, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.00096-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

23S rRNA Nucleotides in the Peptidyl Transferase Center Are Essential for Tryptophanase Operon Induction{triangledown}

Rui Yang,1 Luis R. Cruz-Vera,2 and Charles Yanofsky1*

Department of Biological Sciences, Stanford University, Stanford, California 94305-5020,1 Department of Biological Sciences, University of Alabama-Huntsville, Huntsville, Alabama 358992

Received 24 January 2009/ Accepted 19 March 2009

Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNAPro leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNAPro by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNAPro to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNAPro from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression.

* Corresponding author. Mailing address: Department of Biological Sciences, Stanford University, Stanford, CA 94305. Phone: (650) 725-1835. Fax: (650) 725-8221. E-mail: yanofsky{at}stanford.edu

{triangledown} Published ahead of print on 27 March 2009.

Journal of Bacteriology, June 2009, p. 3445-3450, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.00096-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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