Naslov
Mutational analysis defines the role of conserved residues in the predicted catalytic pocket and suggests a model of protein-RNA interactions of the rRNA:m6A methyltransferase ErmC'

Autori
Maravić, Gordana ; Bujnicki, Janusz M. ; Feder, Marcin ; Pongor, Sandor ; Flögel, Mirna

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
8th Annual Meeting of the RNA Society. Book of abstracts / - Beč, 2003

Skup
8th Annual Meeting of the RNA Society

Mjesto i datum
Beč, Austrija, 01.07.2003. - 06.07.2003

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
methyltransferase ErmC'; site-directed mutagenesis; RNA binding; reaction mechanism; MLS resistance

Sažetak
Erm methyltransferases (MTases) mediate the resistance to the macrolide-lincosamide-streptogramin B antibiotics via dimethylation of a specific adenine residue in 23 S rRNA (A2058 in E.coli). Despite the available structural data and functional analyses on the level of the RNA substrate, the knowledge about the reaction mechanism is still very limited, while the substrate-binding site and residues involved in RNA recognition by the Erm MTases remain completely unknown. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC' and to identify the area of protein-RNA interactions we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m6A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m6A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. Furthermore, our results suggest that the key RNA-binding residues are located not in the small domain, but in the large catalytic domain, facing the cleft between the two domains. Based on the mutagenesis data, we were able to construct the preliminary 3D model of ErmC' complexed with the minimal substrate. The identification of the RNA-binding site of ErmC' may be useful for structure-based design of novel drugs that specifically block the substrate-binding site of Erm MTases.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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