engleski

A new approach to in vitro analysis of 16S rRNA methyltransferases interaction with small ribosomal subunit

Methyltransferases play important role in ribosome biogenesis and decorate rRNA with methyl groups during different stages of ribosome subunits maturation. However, rRNA methyltransferases can also have critical importance for antibiotic resistance, and one the emerging mechanisms of resistance to aminoglycoside antibiotics is the methylation of 16S rRNA in the decoding center of small ribosomal subunit. 16S rRNA methyltransferases conferring resistance to aminoglycoside antibiotics can be found in antibiotic producers, as well as resistant pathogenic strains found in clinical isolates, and represent a major health concern in the fight against aminoglycoside antibiotic resistance. In vitro study of 16S rRNA methyltransferases is hampered by the fact that 16S rRNA methyltransferases responsible for resistance against aminoglycoside antibiotics act upon mature, fully assembled 30S ribosomal subunits as the substrate. Their activity cannot be assayed on naked 16S rRNA or oligonucleotides, because metyltransferases make intricate network of interactions with fully folded 16S rRNA in the assembled 30S ribosomal subunits. We were interested in developing an assay for direct in vitro detection and analysis of macromolecular interactions between these small methylating enzymes and their large, complex macromolecular interacting partner – the 30S ribosomal subunits. First, a simple and qualitative assay for detection of the macromolecular complex between 16S rRNA methyltransferases and small ribosomal subunit has been developed, based on pull-down assay, revealing for the first time that the macromolecular complexes between methyltransferases and 30S ribosomal subunit are stable enough to be physically isolated. Furthermore, the binding of several methyltransferases from both antibiotic producing (Sgm, KamB) and resistant pathogenic strains (RmtA, RmtC, and NpmA) has been quantitatively analyzed by a novel technique, called microscale thermophoresis. The dissociation constants for the methyltransferase complexes with small ribosomal subunits have been determined, and they were in micromolar range. Thus, we have shown that the in vitro analysis of methyltransferase binding to ribonucleoprotein particles, such as small ribosomal subunits, is feasible. The results of the in vitro analysis of 16S rRNA methyltransferase binding to immature small ribosomal subunits will be presented and the timing of 16S rRNA methylation during the late phases of 30S ribosomal subunit maturation will be discussed.

methyltransferase ; ribosome ; antibiotic resistance ; microscale thermophoresis

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