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. 1999 Mar 19;287(1):33-45.
doi: 10.1006/jmbi.1999.2600.

The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre

B T Porse  1 , E Cundliffe, R A Garrett
Affiliations

The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre

B T Porse et al. J Mol Biol. .

Abstract

Micrococcin-resistant mutants of Bacillus megaterium that carry mutations affecting ribosomal protein L11 have been characterised. The mutants fall into two groups. "L11-minus" strains containing an L11 gene with deletions, insertions or nonsense mutations which grow 2.5-fold slower than the wild-type strain, whereas other mutants carrying single-site substitutions within an 11 amino acid residue segment of the N-terminal domain of L11 grow normally. Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which regulates GTP hydrolysis on ribosomal factors. Micrococcin binding within the rRNA component of this centre was probed on wild-type and mutant ribosomes, in vivo, using dimethyl sulphate where it generated an rRNA footprint indistinguishable from that produced in vitro, even after the cell growth had been arrested by treatment with either kirromycin or fusidic acid. No drug-rRNA binding was detected in vivo for the L11-minus mutants, while reduced binding (approximately 30-fold) was observed for two single-site mutants P23L and P26L. For the latter, the reduced drug affinity alone did not account for the resistance-phenotype because rapid cell growth occurred even at drug concentrations that would saturate the ribosomes. Micrococcin was also bound to complexes containing an rRNA fragment and wild-type or mutant L11, expressed as fusion proteins, and they were probed with proteinases. The drug produced strong protection effects on the wild-type protein and weak effects on the P23L and P26L mutant proteins. We infer that inhibition of cell growth by micrococcin, as for thiostrepton, results from the imposition of a conformational constraint on protein L11 which, in turn, perturbs the function(s) of the ribosomal factor-guanosine nucleotide complexes.

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