Puglisi J D, Pütz J, Florentz C, Giegé R
UPR Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
Nucleic Acids Res. 1993 Jan 11;21(1):41-9. doi: 10.1093/nar/21.1.41.
Mutations have been designed that disrupt the tertiary structure of yeast tRNA(Asp). The effects of these mutations on both tRNA structure and specific aspartylation by yeast aspartyl-tRNA synthetase were assayed. Mutations that disrupt tertiary interactions involving the D-stem or D-loop result in destabilization of the base-pairing in the D-stem, as monitored by nuclease digestion and chemical modification studies. These mutations also decrease the specificity constant (kcat/Km) for aspartylation by aspartyl-tRNA synthetase up to 10(3)-10(4) fold. The size of the T-loop also influences tRNA(Asp) structure and function; change of its T-loop to a tetraloop (-UUCG-) sequence results in a denatured D-stem and an almost 10(4) fold decrease of kcat/Km for aspartylation. The negative effects of these mutations on aspartylation activity are significantly alleviated by additional mutations that stabilize the D-stem. These results indicate that a critical role of tertiary structure in tRNA(Asp) for aspartylation is the maintenance of a base-paired D-stem.
已设计出破坏酵母天冬氨酸tRNA（tRNA(Asp)）三级结构的突变。测定了这些突变对tRNA结构以及酵母天冬氨酰-tRNA合成酶特异性天冬氨酰化作用的影响。通过核酸酶消化和化学修饰研究监测发现，破坏涉及D茎或D环的三级相互作用的突变会导致D茎中碱基配对的不稳定。这些突变还会使天冬氨酰-tRNA合成酶天冬氨酰化的特异性常数（kcat/Km）降低高达10³ - 10⁴倍。T环的大小也会影响tRNA(Asp)的结构和功能；将其T环变为四环（-UUCG-）序列会导致D茎变性，且天冬氨酰化的kcat/Km降低近10⁴倍。稳定D茎的额外突变可显著减轻这些突变对天冬氨酰化活性的负面影响。这些结果表明，tRNA(Asp)三级结构对天冬氨酰化的关键作用是维持碱基配对的D茎。
