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Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase

Published online by Cambridge University Press:  07 July 2001

ALLAN K. NIELSEN ,
STEPHEN DOUTHWAITE  and
BIRTE VESTER
ALLAN K. NIELSEN
Affiliation:
Department of Molecular Biology, University of Odense, DK-5230 Odense M, Denmark Present address: Department of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark.
STEPHEN DOUTHWAITE
Affiliation:
Department of Molecular Biology, University of Odense, DK-5230 Odense M, Denmark
BIRTE VESTER
Affiliation:
RNA Regulation Centre, Department of Molecular Biology, University of Copenhagen, DK-1307 Copenhagen K, Denmark
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Abstract

Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S rRNA and the adjacent single-stranded region around A2058. An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase. Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within the 72-mer RNA. The RNAs were passed through a series of rounds of methylation with ErmE. After each round, RNAs were selected that had partially or completely lost their ability to be methylated. After several rounds of methylation/selection, 187 subclones were analyzed. Forty-three of the subclones contained substitutions at single sites, and these are confined to 12 nucleotide positions. These nucleotides, corresponding to A2051–A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase. The structure formed by these nucleotides is highly conserved throughout bacterial rRNAs, and is proposed to constitute the motif that is recognized by all the Erm methyltransferases.

Keywords

MLS resistancenegative in vitro selectionprotein–RNA interactionrRNA modification
Type
Research Article
Information
RNA , Volume 5 , Issue 8 , August 1999 , pp. 1034 - 1041
Copyright
1999 RNA Society

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