Modified Uridines with C5-methylene Substituents at the First Position of the tRNA Anticodon Stabilize U·G Wobble Pairing during Decoding

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 (m 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 (⌬mnmE) 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 U⅐G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing m 5 U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the m 5 U⅐G base pair does not have classical U⅐G wobble geometry. These structures provide help to explain how the m 5 U modification enables efficient decoding of UUG codons.The genetic code is deciphered by the anticodons of tRNAs, which carry an amino acid at the 3Ј end, bind to a specific codon in the mRNA, and transfer their amino acid to the growing polypeptide chain on the ribosome. In codon-anticodon interactions in the ribosome, the second and third bases (positions 35 and 36) of the anticodon base pair with the second and first bases of the codon, respectively, following Watson-Crick (WC) 2 -type pairing rules. Structural studies of the 30 S ribosomal subunit revealed that the conserved bases A1492, A1493, and G530 in the decoding center of the 16 S rRNA specifically monitor these two WC-type pairings by A-minor interactions (1, 2). These interactions induce a large conformational rearrangement of the 30 S subunit that is necessary for tRNA selection and maintaining decoding fidelity. In contrast, base-pairing between the first base of the anticodon (position 34) and the third base of the codon does not always conform to WC-type pairing rules, so that synonymous codons for an amino acid are deciphered by a minimum set of tRNA anticodons. This behavior is referred to as "wobble pairing" (3), which is a highly evolved system required for the degeneracy of the genetic code by which 61 sense codons are deciphered into 20 amino acids by a limited set of tRNA species. Modified nucleosides are often found at the wobble position of tRNA anticodons (4 -8). The wobble modifications play critical roles in modulating codon recognition by restricting, e...

Celebrating wobble decoding: Half a century and still much is new

“…For instance, pseudouridine at position 32 uses a water mediated base-backbone interaction to stabilize the interaction between nucleosides 32 and 38. 138 While a stronger interaction, like that of a canonical base pair at this position could lead to a loss in flexibility of the loop domain, a weaker interaction could result in loss of stacking in the ASL resulting in a disruption of the functional U-turn conformation of the ASL. Modifications at the 32 nd and 38 th positions of the ASL may be important as a possible means of modulating the interaction between the two nucleosides and base stacking in the loop.…”

Taurine-containing Uridine Modifications in tRNA Anticodons Are Required to Decipher Non-universal Genetic Codes in Ascidian Mitochondria

“…Furthermore, it has been shown that the m 5 U modification in human mt tRNA Leu (UUR) stabilizes the m 5 U:G wobble pairing at the ribosomal A site during decoding (29). A structural study of the ribosomal 30S subunit containing codon-anticodon pairing at the A-site provided a mechanistic explanation for the manner in which the 5-taurinomethyl modification enables efficient decoding of G-ending codons (30). In the human mitochondrial decoding system, m 5 (s 2 )U is found in five tRNAs for Leu(UUR), Trp, Lys, Glu, and Gln (23,24,31,32).…”

Clinical significance of taurine

“…One reaction involves the detoxification of hypochlorous acid by taurine resulting in the formation of taurine chloramine, a product that has the added benefit of possessing anti-inflammatory activity (Schuller-Levis and Park 2003;Marcinkiewicz and Kontny 2013;Kim and Cha 2014). Taurine also forms a conjugate (5-taurinomethyluridine) with the wobble position uridine of tRNA Leu(UUR) , a reaction that enhances the interaction between the modified uridine and guanine (Kirino et al 2005;Kurata et al 2008;Schaffer et al 2013). Biochemically, this enhances the binding of the UUG codon to the taurine-modified AAU anticodon of tRNA Leu(UUR) , facilitating UUG decoding.…”

“…These fundamental biochemical events are defective in the mitochondrial disease, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), which is caused by one of several tRNA Leu(UUR) mutations. One of the major biochemical abnormalities of the tRNA Leu(UUR) mutations is decreased formation of 5-taurinomethyluridine in the wobble position of tRNALeu(UUR) (Kirino et al 2005;Kurata et al 2008). Because taurine conjugation enhances the interaction of the UUG codon with the taurine-modified AAU anticodon of tRNA Leu(UUR) , the synthesis of mitochondria encoded proteins, such as ND6, is highly dependent upon the 5-taurinomethyluridine modification (Jong et al 2012).…”