Abstract
Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (taum(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (DeltamnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U.G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing taum(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the taum(5)U.G base pair does not have classical U.G wobble geometry. These structures provide help to explain how the taum(5)U modification enables efficient decoding of UUG codons.
Highlights
JULY 4, 2008 VOLUME 283 NUMBER 27 provide help to explain how the m5U modification enables efficient decoding of UUG codons
We previously found that mitochondrial tRNAsLeu(UUR) with pathogenic point mutations isolated from MELAS patients lacked the 5-taurinomethyluridine (m5U) modification and caused a decoding defect
Biochemical studies using an in vitro mitochondrial translation system revealed that the wild type tRNALeu(UUR) whose m5U modification was surgically replaced by an unmodified uridine exhibited severely reduced UUG decoding but no decrease in UUA decoding [27]
Summary
Construction of E. coli tRNALeu(UUR) Bearing the Wobble Modification—The cmnm5U and m5U nucleosides were chemically synthesized [21, 28, 29], and 5Ј- and 3Ј(2Ј)-diphosphorylation of these nucleosides was performed as previously described [30]. Four different amounts (0.25, 0.5, 0.75, and 1 pmol) of 5Ј-32P-labeled E. coli tRNAsLeu(UUR) with or without wobble modifications in a mixture (10 l) consisting of 50 mM Tris-HCl (pH 7.5), 6.5 mM MgCl2, 60 mM KCl, 1 mM DTT, and 2 mM spermine were added to the ribosomal mixtures, and a nonenzymatic binding reaction was performed at 37 °C for 12 min. TRNAsLeu(UUR) with or without wobble modifications were leucylated at 37 °C for 10 min in a reaction mixture (30 l) consisting of 100 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 2 mM ATP, 20 mM KCl, 1 mM DTT, 20% dimethyl sulfoxide, 100 M [14C]L-leucine, and 1 g/l E. coli leucyl-tRNA synthetase. These results suggest that compare it with the m5U1⁄7G base pair [52]
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