Behlen L S, Sampson J R, DiRenzo A B, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1990 Mar 13;29(10):2515-23. doi: 10.1021/bi00462a013.
Yeast tRNA(Phe) lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and G18 at a rate very similar to that of its fully modified counterpart. The rates of cleavage for 28 tRNA(Phe) mutants were determined to define the structural requirements of this reaction. The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography. Nucleotide changes that disrupted the tertiary interactions of tRNAPhe reduced the rate of cleavage even when they were distant from the lead binding pocket. However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction of a useful probe for tRNA(Phe) structure. Certain mutants resulted in the enhancement of cleavage at a "cryptic" site at C48. The sequences of Escherichia coli tRNA(Phe) and yeast tRNA(Arg) were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage. This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions.
缺乏修饰核苷酸的酵母tRNA（Phe）在核苷酸U17和G18之间发生铅催化切割的速率与其完全修饰的对应物非常相似。测定了28个tRNA（Phe）突变体的切割速率，以确定该反应的结构要求。发现切割速率非常依赖于X射线晶体学定义的两个铅配位嘧啶的身份和正确定位。即使tRNAPhe的三级相互作用被破坏的核苷酸变化远离铅结合口袋，也会降低切割速率。然而，设计用于维持三级相互作用的核苷酸变化显示出正常的切割速率，从而使该反应成为tRNA（Phe）结构的有用探针。某些突变体导致在C48的“隐蔽”位点处切割增强。改变了大肠杆菌tRNA（Phe）和酵母tRNA（Arg）的序列，使其获得了在U17处切割的能力，证实了我们对切割结构要求的理解。对铅切割结构域的这种诱变分析为自催化自我切割反应的类似分析提供了有用的指导。
