The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase
- 1Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
- 2Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
- Corresponding author: rfahlman{at}ualberta.ca
Abstract
Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNALeu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNALeu (CAG)—a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.
Keywords
- N-end rule
- quantitative mass spectrometry
- L/F transferase
- aminoacyl-tRNA protein transferase
- tRNA recognition
- isoacceptors
Footnotes
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Abbreviations: Aminoacyl-tRNA, aminoacylated tRNA; L/F transferase, leucyl/phenylalanyl tRNA protein transferase; ATE1, arginine tRNA protein transferase 1; MALDI-ToF MS, matrix assisted laser desorption/ionization time-of-flight mass spectrometry; LeuRS, leucyl-tRNA synthetase; PheRS, phenylalanyl-tRNA synthetase; MetRS, methionyl-tRNA synthetase; EF-Tu, elongation factor thermal unstable; rA-aa, aminoacyl adenosine; rA-Phe, phenylalanyl adenosine
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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.044529.114.
- Received January 23, 2014.
- Accepted April 28, 2014.
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