Hoburg A, Aschhoff H J, Kersten H, Manderschied U, Gassen H G
J Bacteriol. 1979 Nov;140(2):408-14. doi: 10.1128/jb.140.2.408-414.1979.
To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TUC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNA(f) (Met) the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the V(max) value is decreased by one-half. (ii) tRNA(f) (Met) species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNA(f) (Met) species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNA(t) (Met) species, however, differ from each other: "Met-tRNA(f) (Met) rT" is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA(1) (Phe) and tRNA(2) (Phe), were obtained from B. subtilis. tRNA(1) (Phe) accumulates only under special growth conditions and is an incompletely modified precursor oftRNA(2) (Phe): in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N(6)-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU(3)-directed formation of fMet-Phe, the undermodified tRNA(1) (Phe) is always less efficient at Mg(2+) concentrations between 5 and 15 mM than its mature counterpart.
为阐明转移核糖核酸（tRNA）修饰在蛋白质合成中的细微功能，从枯草芽孢杆菌制备了在特定位置修饰不同的成对tRNA。这些tRNA在反密码子环、额外臂和TUC环的修饰上存在差异。在程序化核糖体上，对这些tRNA种类在氨酰化以及起始和肽键形成方面的功能特性进行了比较。这些实验证明了以下几点。（i）在tRNA(f)（Met）中，来自大肠杆菌的7-甲基鸟苷形成酶将额外臂中的鸟苷46甲基化为7-甲基鸟苷，这改变了枯草芽孢杆菌甲硫氨酰-tRNA合成酶的氨酰化动力学。在重复实验中，V(max)值降低了一半。（ii）在位置54为核糖胸苷（rT54）或尿苷（U54）的tRNA(f)（Met）种类，分别从未经处理或经甲氧苄啶处理的枯草芽孢杆菌中获得。分别带有U54和rT54的甲酰化fMet-tRNA(f)（Met）种类，在含有AUG、起始因子和70S核糖体的体外起始系统中功能相同。然而，未甲酰化的Met-tRNA(t)（Met）种类彼此不同：“Met-tRNA(f)（Met）rT”无活性，而U54对应的种类能有效形成起始复合物。（iii）从枯草芽孢杆菌中获得了两种同功受体tRNA(1)（Phe）和tRNA(2)（Phe）。tRNA(1)（Phe）仅在特殊生长条件下积累，是tRNA(2)（Phe）的不完全修饰前体：在反密码子的第一位，鸟苷取代了Gm，在反密码子3'端旁边，（异戊烯基）腺苷取代了2-硫甲基-N(6)-（异戊烯基）腺苷。两种tRNA在氨酰化动力学方面表现相同。在依赖因子的AUGU(3)指导的fMet-Phe形成过程中，在5至15 mM的Mg(2+)浓度下，修饰不足的tRNA(1)（Phe）在效率上总是低于其成熟对应物。
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