Yarian C, Marszalek M, Sochacka E, Malkiewicz A, Guenther R, Miskiewicz A, Agris P F
Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
Biochemistry. 2000 Nov 7;39(44):13390-5. doi: 10.1021/bi001302g.
Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.
核苷修饰对所有tRNA的结构都很重要，对某些tRNA种类的功能也至关重要。具有UUU反密码子的人tRNA(Lys3)(UUU)转录本以及相应的反密码子茎环结构域（ASL(Lys3)(UUU)）无法与聚腺苷酸编程核糖体结合。为了确定tRNA(Lys)种类的特定反密码子结构域修饰核苷是否会恢复核糖体结合并影响热稳定性，我们化学合成了ASL(Lys)十七聚体，并位点特异性地掺入了反密码子结构域修饰核苷假尿苷（Ψ(39)）、5-甲基氨基甲基尿苷（mnm(5)U(34)）和N6-苏氨甲酰腺苷（t(6)A(37)）。t(6)A(37)和mnm(5)U(34)的掺入为反密码子环贡献了结构，表现为ΔS增加，并显著增强了ASL(Lys3)(UUU)结合聚腺苷酸编程核糖体的能力。ASL(Lys3)(UUU)-t(6)A(37)和ASL(Lys3)(UUU)-mnm(5)U(34)都不能结合AAG编程核糖体。只有同时存在t(6)A(37)和mnm(5)U(34)才能使ASL(Lys3)(UUU)结合AAG编程核糖体，并将其对聚腺苷酸编程核糖体的亲和力提高到天然大肠杆菌tRNA(Lys)的水平。具有摆动位置34为C的人tRNA(Lys1,2)(CUU)的完全未修饰的反密码子茎环结合AAG，但不会摆动到AAA，即使ASL用t(6)A(37)修饰。数据表明，tRNA(Lys)(UUU)种类需要环中的反密码子结构域修饰来为反密码子赋予有序结构以与AAA进行核糖体结合，并且需要修饰核苷的组合来结合AAG。
