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Nucleic Acids Research 2004 32(22):6548-6556; doi:10.1093/nar/gkh1001
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Published online 14 December 2004

Nucleic Acids Research, Vol. 32 No. 22 © Oxford University Press 2004; all rights reserved

A 16S rRNA–tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome

Wayne Huggins and Paul Wollenzien*

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA

* To whom correspondence should be addressed. Tel: +1 919 515 5703; Fax: +1 919 515 2047; Email: paul_wollenzien{at}ncsu.edu

Received September 10, 2004; Revised and Accepted November 26, 2004

Ribosome complexes containing deacyl- or biotinylvalyl- and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo5U nucleoside at position 34 in the anticodon loop. The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo5U34) characterized already by Ofengand and his collaborators [Prince et al. (1982) Proc. Natl Acad. Sci. USA, 79, 5450–5454]. However, in complexes containing deacyl-, an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m5C967 of 16S rRNA and cmo5U34 of the tRNA. Puromycin treatment of the biotinylvalyl- –70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer. These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state.


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W. Huggins, T. Shapkina, and P. Wollenzien
Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s4U8 C13 photocrosslinking
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