Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin.
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 SRL cleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocin binds to a 27-mer SRL model substrate (designated wild-type SRL) via electrostatic interactions to form a nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-type substrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atom at the scissile phosphate, reacts at a fully diffusion-limited rate (k2/K1/2 = 1.1 x 10(9) M-1 s-1). At pH 7.4, the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocin recognizes 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 at least 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRL recognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-G motif 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 revealed that restrictocin exhibits a 10(3)-10(6)-fold smaller ribonuclease activity against single-stranded RNA than do the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findings show how structural features of the SRL substrate facilitate catalysis and provide a mechanism for distinguishing between cognate and noncognate RNA.
Korennykh
Alexei V
AV
Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.
Plantinga
Matthew J
MJ
Correll
Carl C
CC
Piccirilli
Joseph A
JA
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004722:Endoribonucleases / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry* / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005656:Fungal Proteins; D012331:RNA, Fungal; C014801:alpha-sarcin; C036196:MITF protein, Aspergillus restrictus; D004722:Endoribonucleases; D012260:Ribonucleases