Atom-specific mutagenesis reveals structural and catalytic roles for an active-site adenosine and hydrated Mg(2+) in pistol ribozymes Journal Article
| Authors: | Neuner, S.; Falschlunger, C.; Fuchs, E.; Himmelstoss, M.; Ren, A.; Patel, D. J.; Micura, R. |
| Article Title: | Atom-specific mutagenesis reveals structural and catalytic roles for an active-site adenosine and hydrated Mg(2+) in pistol ribozymes |
| Abstract: | The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53–U54 cleavage site. While the N1 of G40 is within 3.4 Å of the modeled G53 2′-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid–base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2′-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg2+ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg2+ playing a catalytic role in cleavage. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim |
| Keywords: | rna; crystal structure; catalysis; biomolecules; hydration; mutagenesis; structure-function relationship; oligonucleotides; biological functions; nucleosides; rna modification; ribozymes; acid-base catalysis; structure–function relationship; guns (armament); bridging units; h-bond network; rna modifications; crystal atomic structure |
| Journal Title: | Angewandte Chemie - International Edition |
| Volume: | 56 |
| Issue: | 50 |
| ISSN: | 1433-7851 |
| Publisher: | Wiley Blackwell |
| Date Published: | 2017-12-11 |
| Start Page: | 15954 |
| End Page: | 15958 |
| Language: | English |
| DOI: | 10.1002/anie.201708679 |
| PROVIDER: | scopus |
| PUBMED: | 29098759 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 2 January 2018 -- Source: Scopus |
