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Efficient substrate cleavage catalyzed by hammerhead ribozymes derivatized with selenium for X-ray crystallography.

Abstract：

: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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Brandt G,Carrasco N,Huang Z

doi

10.1021/bi060455m

subject

Has Abstract

pub_date

2006-07-25 00:00:00

pages

8972-7

issue

29

eissn

0006-2960

issn

1520-4995

journal_volume

45

pub_type

杂志文章

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