Over Expression of a tRNA Leu Isoacceptor Changes Charging Pattern of Leucine tRNAs and Reveals New Codon Reading Michael A. Sørensen 1 †, Johan Elf 2 †, Elli Bouakaz 2 †, Tanel Tenson 3 Suparna Sanyal 2 , Glenn R. Bjo ¨ rk 4 and Ma ˚ ns Ehrenberg 2 * 1 Department of Molecular Cell Biology, University of Copenhagen, DK-1353 Copenhagen, Denmark 2 Department of Cell and Molecular Biology, Uppsala University, BMC, 75124 Uppsala, Sweden 3 Institute of Technology, Tartu University, Tartu 51010 Estonia 4 Department of Molecular Biology, Umea ˚ University 90187 Umea ˚, Sweden During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNA Leu GAG . As predicted, the charged level of tRNA Leu GAG increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNA Leu UAG increased unexpectedly. To remove this apparent incon- sistency between theory and experiment we postulated a previously unknown common codon for tRNA Leu GAG and tRNA Leu UAG . Subsequently, we demonstrated that the tRNA Leu GAG codon CUU is, in fact, read also by tRNA Leu UAG , due to a uridine-5-oxyacetic acid modification. q 2005 Elsevier Ltd. All rights reserved. Keywords: aminoacylation; systems biology; decoding; staruation; kinetics *Corresponding author Introduction Cells use different transfer RNA (tRNA) mol- ecules aminoacylated (charged) with the same type of amino acid to decode synonymous codons in messenger RNAs (mRNAs). 40 Recently, it was suggested 1 that the charged levels of such tRNA “isoacceptors” will be selectively reduced in response to limited supply of their cognate amino acid. This prediction follows from intracellular flow balance relations, stating that the rate of amino- acylation of an isoacceptor by its cognate amino- acyl-tRNA synthetase 2 must always be equal to its rate of deacylation in protein synthesis. To a first approximation the rate of aminoacylation of an isoacceptor is proportional to the concentration of its deacylated form. Furthermore, its rate of deacylation in protein synthesis is proportional to the usage frequency of its cognate codon(s) on translating ribosomes. Therefore, the concentration of a deacylated tRNA normalized to the usage frequency of its cognate codons in translation must be equal for all tRNA isoacceptors in the same family. When amino acid limitation becomes increasingly severe, the charged level of the tRNA isoacceptor with the smallest ratio between its total concentration and the usage frequency of its cognate codons in translation will therefore approach zero, while the charged levels of the other tRNAs in the family will stabilize at intermediate values. 1 From this follows also the prediction that the charged fraction of a tRNA isoacceptor, originally at a low level during starvation for its cognate amino acid, will increase significantly when it is over expressed. Accordingly, one way that cells could regulate gene expression during amino acid limitation would be by tuning the relative concentrations of tRNAs in families of isoacceptors. The theory successfully identified the choices of synonymous control codons in attenuation leader sequences in Escherichia coli, 3 assuming that these should be read by isoacceptors for which the charged levels respond most sensitively to amino 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. † M.A.S., J.E. & E.B. contributed equally to this work. Abbreviations used: mRNA, messenger RNA; tRNA; transfer RNA. E-mail address of the corresponding author: ehrenberg@xray.bmc.uu.se doi:10.1016/j.jmb.2005.08.076 J. Mol. Biol. (2005) 354, 16–24