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J. Biol. Chem., Vol. 281, Issue 46, 35235-35244, November 17, 2006

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Involvement of Helix 34 of 16 S rRNA in Decoding and Translocation on the Ribosome^*

Andrew Kubarenko¹, Petr Sergiev, Wolfgang Wintermeyer^¶, Olga Dontsova², and Marina V. Rodnina³

From the Institute of Physical Biochemistry, University of Witten/Herdecke, 58448 Witten, Germany, the Department of Chemistry, M. V. Lomonosov Moscow State University, 119899 Moscow, Russia, and the ^¶Institute of Molecular Biology, University of Witten/Herdecke, 58448 Witten, Germany

Helix 34 of 16 S rRNA is located in the head of the 30 S ribosomal subunit close to the decoding center and has been invoked in a number of ribosome functions. In the present work, we have studied the effects of mutations in helix 34 both in vivo and in vitro. Several nucleotides in helix 34 that are either highly conserved or form important tertiary contacts in 16 S rRNA (U961, C1109, A1191, and A1201) were mutated, and the mutant ribosomes were expressed in the Escherichia coli MC250 7 strain that lacks all seven chromosomal rRNA operons. Mutations at positions A1191 and U961 reduced the efficiency of subunit association and resulted in structural rearrangements in helix 27 (position 908) and helix 31 (position 974) of 16 S rRNA. All mutants exhibited increased levels of frameshifting and nonsense readthrough. The effects on frameshifting were specific in that -1 frameshifting was enhanced with mutant A1191G and +1 frameshifting with the other mutants. Mutations of A1191 moderately (2-fold) inhibited tRNA translocation. No significant effects were found on efficiency and rate of initiation, misreading of sense codons, or binding of tRNA to the E site. The data indicate that helix 34 is involved in controlling the maintenance of the reading frame and in tRNA translocation.

Received for publication, August 22, 2006 , and in revised form, September 21, 2006.

^* This work was supported in part by grants from Howard Hughes Medical Institute, Russian Foundation for Basic Research, and the Ministry of Science and Technology for Leading Scientific Schools (to O. A. D.) and grants from the Deutsche Forschungsgemeinschaft, the Alfried Krupp von Bohlen und Halbach-Stiftung, and the Fonds der Chemischen Industrie (to M. V. R.) 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 Supported by a Young Scientist INTAS Fellowship from the European Commission.

² To whom correspondence may be addressed. Tel.: 7-494-9328824, Fax: 7-495-9393181; E-mail: dontsova{at}genebee.msu.su. ³ To whom correspondence may be addressed. Tel.: 49-2302-926205; Fax: 49-2302-926117; E-mail: rodnina{at}uni-wh.de.

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