Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase

Dennis Reichert, Helena Schepers, Julian Simke, Horst Lechner, Wolfgang Dörner, Birte Höcker, Bart Jan Ravoo, Andrea Rentmeister*

*Korrespondierende/r Autor/in für diese Arbeit

Publikation: Beitrag in einer FachzeitschriftArtikelBegutachtung

Abstract

The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled the manipulation and investigation of many steps in the involved processes. However, examples for light-mediated control of eukaryotic mRNA processing and the responsible enzymes are still rare. In particular, methylation of the 5' cap of mRNA is required for ribosome assembly, and the responsible guanine-N7 methyltransferase (MTase) from E. cuniculi (Ecm1) proved suitable for activating translation. Here, we report on a photoswitchable MTase obtained by bridging the substrate-binding cleft of Ecm1 with a tetra-ortho-methoxy-azobenzene. This azobenzene derivative is characterized by efficient trans-to-cis isomerization using red light at 615 nm. Starting from a cysteine-free Ecm1 variant (ΔCys), we used a computational approach to identify suitable conjugation sites for the azobenzene moiety. We created and characterized the four best-ranked variants, each featuring two appropriately positioned cysteines close to the substrate-binding cleft. Conjugating and crosslinking the azobenzene between C149/C155 in a designed Ecm1 variant (VAR3-Az) enabled light-dependent modulation of the MTase activity and showed a 50% higher activity for the cis form than the trans-form of the azobenzene conjugated to VAR3-Az.

Originalspracheenglisch
Seiten (von - bis)1484-1490
Seitenumfang7
FachzeitschriftRSC Chemical Biology
Jahrgang2
Ausgabenummer5
DOIs
PublikationsstatusVeröffentlicht - 7 Okt 2021

ASJC Scopus subject areas

  • Chemie (sonstige)
  • Molekularbiologie
  • Biochemie, Genetik und Molekularbiologie (sonstige)
  • Biochemie

Fingerprint

Untersuchen Sie die Forschungsthemen von „Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase“. Zusammen bilden sie einen einzigartigen Fingerprint.

Dieses zitieren