Linkage between Substrate Recognition and Catalysis during Cleavage of Sarcin/Ricin Loop RNA by Restrictocin

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• 作者：Alexei V. Korennykh ; Matthew J. Plantinga ; Carl C. Correll ; Joseph A. Piccirilli
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 6, 2007
• 年：2007
• 卷：46
• 期：44
• 页码：12744 - 12756
• 全文大小：598K
• 年卷期：v.46,no.44(November 6, 2007)
• ISSN：1520-4995

文摘

Restrictocin is a site-specific endoribonuclease that inactivates ribosomes by cleaving the sarcin/ricin loop (SRL) of 23S-28S rRNA. Here we present a kinetic and thermodynamic analysis of the SRLcleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocinbinds to a 27-mer SRL model substrate (designated wild-type SRL) via electrostatic interactions to forma nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-typesubstrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atomat the scissile phosphate, reacts at a fully diffusion-limited rate (k₂/K_1/2 = 1.1 × 10⁹ M^-1 s^-1). At pH 7.4,the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocinrecognizes the SRL structure, which imparts 4.3 kcal/mol transition state stabilization relative to a single-stranded RNA. The two conserved SRL modules, bulged-G motif and GAGA tetraloop, contribute atleast 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRLrecognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-Gmotif to progress from the nonspecific ground state complex (E:S) to the higher-energy-specific complex(E·S) en route to the chemical transition state. Comparison of restrictocin with other ribonucleases revealedthat restrictocin exhibits a 10³-10⁶-fold smaller ribonuclease activity against single-stranded RNA thando the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findingsshow how structural features of the SRL substrate facilitate catalysis and provide a mechanism fordistinguishing between cognate and noncognate RNA.