Modified Uridines, Contributors of Novel Chemistries to Functional RNA Structures

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9986011AgrisRNAs contain the standard nucleosides adenosine, uridine, guanosine and cytosine. In addition, RNA contains some 100 naturally occurring modifications of the four major nucleosides. The modifications are as simple as methylations and thiolations and as complex as amino acid derivatives and tricylic additions. All are enzymatically synthesized after synthesis (transcription) of the RNA. Transfer RNAs (tRNA) having a large number and variety of modified nucleosides, its structure and function readily assayed, is a good model RNA for the study of modified nucleoside contributions to structure and function. Conventional wisdom has led researchers to believe that tRNA, responsible for bringing individual amino acids to the ribosome in response to genetic coding triplets (codons) in the messenger RNA (mRNA), do not require modified nucleosides to function in protein synthesis. However, this laboratory has recently determined that at least two modified nucleosides, 2-thiouridine derivatives (s2U) at "wobble" position-34 of and 6-threonylcarbamoyladenosine (t6A) at position-37 of tRNA's anticodon stem and loop domain (ASL), are individually capable of restoring ribosome binding to the otherwise inactive ASL of human lysine-3 tRNA. Position-34 is the first nucleoside of the anticodon that binds its complementary codon in the mRNA on the ribosome. Thiouridines occur in tRNAs for glutamine, glutamic acid and lysine; t6A occurs in tRNAs for lysine. Techniques for site-specific introduction of modified and stable isotope labeled nucleosides developed in this laboratory make it possible to probe the contributions these nucleosides alone and together, and in combination with other modifications provide to RNA function and structure. The project's long-term objective continues to be an elucidation of the physicochemical contributions of modified uridines to the biological functions of RNA. Several testable hypotheses have been formulated: a) Thiouridine-34 dependent ribosome binding is common to tRNAs with uridine-rich anticodon loops and is important for A-site, as well as P-site binding. b) Whereas t6A37 restores ribosomal binding to the ASLLys3UUU, the commonly occurring position-37 modification of glutamine and glutamic acid tRNAs, 2-methyladenosine-37 (m2A37), will not restore binding to ASLGlnSUC and ASLGluSCU. c) Modification-dependent ribosome binding is sequence context dependent. d) Anticodon stem and loop modifications provide optimum ribosomal binding architecture to lysine, glutamine and glutamic acid tRNAs by significantly influencing chemical and conformational dynamics of the ASL. Biochemical and molecular biology techniques will be used as approaches, including the use of quantitative P- and A-site ribosomal binding, and quantitative footprinting of tRNA on ribosomal P- and A-site 16S rRNA nucleosides. The physicochemical contributions of modifications (thermal stability, base stacking interactions and structure) will be related to their abilities to restore ribosome binding to lysine, glutamine and glutamic acid ASLs. Effects of modification on ribosome binding and footprinting will be related to the modifications' effects on stability, structure and dynamics.