Efficient substrate cleavage catalyzed by hammerhead ribozymes derivatized with selenium for X-ray crystallography.
Because oxygen and selenium are in the same group (Family VI) in the periodic table, the site-specific mutagenesis at the atomic level by replacing RNA oxygen with selenium can provide insights on the structure and function of catalytic RNAs. We report here the first Se-derivatized ribozymes transcribed with all nucleoside 5'-(alpha-P-seleno)triphosphates (NTPalphaSe, including A, C, G, and U). We found that T7 RNA polymerase recognizes NTPalphaSe Sp diastereomers as well as the natural NTPs, whereas NTPalphaSe Rp diastereomers are neither substrates nor inhibitors. We also demonstrated the catalytic activity of these Se-derivatized hammerhead ribozymes by cleaving the RNA substrate, and we found that these phosphoroselenoate ribozymes can be as active as the native one. These hammerhead ribozymes site-specifically mutagenized by selenium reveal the close relationship between the catalytic activities and the replaced oxygen atoms, which provides insight on the participation of oxygen in catalysis or intramolecular interaction. This demonstrates a convenient strategy for the mechanistic study of functional RNAs. In addition, the active ribozymes site-specifically derivatized by selenium will allow for convenient MAD phasing in X-ray crystal structure studies.
Brandt
Gary
G
Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, USA.
Carrasco
Nicolas
N
Huang
Zhen
Z
D018360:Crystallography, X-Ray / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D043264:Phosphodiesterase I / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D018036:Selenium Compounds / Q000737:chemistry*; D012910:Snake Venoms / Q000201:enzymology; D014764:Viral Proteins / Q000378:metabolism
D009711:Nucleotides; D016337:RNA, Catalytic; D018036:Selenium Compounds; D012910:Snake Venoms; D014764:Viral Proteins; C427204:hammerhead ribozyme; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom