New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome
- 1Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA
- 2Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA
- 3Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
Abstract
Three new photoreactive tRNA derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. In two of these derivatives, the 3′ adenosine of yeast tRNAPhe has been replaced by either 2-azidodeoxyadenosine or 2-azido-2′-O-methyl adenosine, while in a third the 3′-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNAPhe and can be cross-linked to components of the 50S ribosomal subunit by irradiation with near-UV light. Characteristic differences in the cross-linking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the peptidyl transferase center.
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Footnotes
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Reprint requests to: Robert A. Zimmermann, Department of Biochemistry and Molecular Biology, University of Massachusetts, 710 North Pleasant Street, Amherst, MA 01003, USA; e-mail: zimmermann{at}biochem.umass.edu; fax: (413) 545-3291.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.425907.
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- Received December 5, 2006.
- Accepted February 12, 2007.
- Copyright © 2007 RNA Society
