Sweta Vangaveti1, William A Cantara2, Jessica L Spears2, Hasan DeMirci3, Frank V Murphy4, Sri V Ranganathan1, Kathryn L Sarachan5, Paul F Agris6. 1. The RNA Institute, University at Albany-SUNY, 1400 Washington Ave., Albany, NY, 12222, USA. 2. Department of Biological Sciences, University at Albany-SUNY, 1400 Washington Ave., Albany, NY, 12222, USA. 3. PULSE Institute, Stanford University, 2575 Sand Hill Road, MS 59, Menlo Park, CA, 94025, USA; Department of Molecular Biology and Genetics, Koc University, Rumelifeneri yolu, Sariyer, Istanbul, 34450, Turkey. 4. Argonne National Laboratories, NE-CAT/Cornell University, 9700 S. Cass Avenue, Lemont, IL, 60439, USA. 5. Department of Biological Sciences, University at Albany-SUNY, 1400 Washington Ave., Albany, NY, 12222, USA; The RNA Institute, University at Albany-SUNY, 1400 Washington Ave., Albany, NY, 12222, USA. Electronic address: kathryn.sarachan@wilson.edu. 6. Department of Biological Sciences, University at Albany-SUNY, 1400 Washington Ave., Albany, NY, 12222, USA; The RNA Institute, University at Albany-SUNY, 1400 Washington Ave., Albany, NY, 12222, USA. Electronic address: paul.agris@duke.edu.
Abstract
Three of six arginine codons (CGU, CGC, and CGA) are decoded by two Escherichia coli tRNAArg isoacceptors. The anticodon stem and loop (ASL) domains of tRNAArg1 and tRNAArg2 both contain inosine and 2-methyladenosine modifications at positions 34 (I34) and 37 (m2A37). tRNAArg1 is also modified from cytidine to 2-thiocytidine at position 32 (s2C32). The s2C32 modification is known to negate wobble codon recognition of the rare CGA codon by an unknown mechanism, while still allowing decoding of CGU and CGC. Substitution of s2C32 for C32 in the Saccharomyces cerevisiae tRNAIleIAU anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of s2C at position 32 is a generalizable property. X-ray crystal structures of variously modified ASLArg1ICG and ASLArg2ICG constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a C32-A38 cross-loop interaction but failed to fully explain the means by which s2C32 restricts I34 wobbling. Computational studies revealed that the adoption of a spatially broad inosine-adenosine base pair at the wobble position of the codon cannot be maintained simultaneously with the canonical ASL U-turn motif. C32-A38 cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site.
Three of six arginine codons (pan class="Chemical">CGU, CGC, and CGA) are decoded by two Escherichia coli tRNAArg isoacceptors. The anticodon stem and loop (ASL) domains of tRNAArg1 and tRNAArg2 both contain inosine and 2-methyladenosine modifications at positions 34 (I34) and 37 (m2A37). tRNAArg1 is also modified from cytidine to 2-thiocytidine at position 32 (s2C32). The s2C32 modification is known to negate wobble codon recognition of the rare CGA codon by an unknown mechanism, while still allowing decoding of CGU and CGC. Substitution of s2C32 for C32 in the Saccharomyces cerevisiae tRNAIleIAU anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of s2C at position 32 is a generalizable property. X-ray crystal structures of variously modified ASLArg1ICG and ASLArg2ICG constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a C32-A38 cross-loop interaction but failed to fully explain the means by which s2C32 restricts I34 wobbling. Computational studies revealed that the adoption of a spatially broad inosine-adenosine base pair at the wobble position of the codon cannot be maintained simultaneously with the canonical ASL U-turn motif. C32-A38 cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site.
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