Probing RNA Structure and Ligand Binding Sites on RNA by Fenton Cleavage

Heidrich, Corina G.; Berens, Christian

doi:10.1002/9783527647064.ch15

Cited by 1 publication

(3 citation statements)

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“…Previously, this approach successfully mapped TET binding sites on the Tet repressor protein TetR [29] and the TET efflux protein TetA [30], as well as TET and TGC binding sites on the ribosome [15]. It is important to note that the Fe 2+ and Mg 2+ in the TET-chelate complex are considered isostructural [31] as Fe 2+ can substitute for Mg 2+ to induce the Tet repressor [29]. Using this approach, we probed empty ribosomes from E. coli CAN/20-E12 (2 µM) in the presence of TET, TGC, and OMC (1 to 125 µM) (Figure 3 and Figure S1).…”

Section: Resultsmentioning

confidence: 99%

“…The rRNA was isolated as described [31]. The ethanol-precipitated pellets were resuspended in 200 μL RE-buffer (300 mM sodium-acetate; 0.5% ( w / v ) sodium dodecyl sulfate (SDS); 5 mM EDTA), supplemented with 8 Units RNAse Inhibitor (40 U/μL; Roche) and stored at 4 °C until use.…”

Section: Methodsmentioning

confidence: 99%

“…The primer extension reaction was performed as described [31]. Briefly, 2 μL of the isolated rRNA (0.5–0.7 µg/μL) was mixed with 1 μL 5× Q-solution (Qiagen, One-step RT-PCR Kit) and 1 μL 5′-[ 32 P]-labelled primer.…”

Section: Methodsmentioning

confidence: 99%

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The Novel Aminomethylcycline Omadacycline Has High Specificity for the Primary Tetracycline-Binding Site on the Bacterial Ribosome

et al. 2016

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Omadacycline is an aminomethylcycline antibiotic with potent activity against many Gram-positive and Gram-negative pathogens, including strains carrying the major efflux and ribosome protection resistance determinants. This makes it a promising candidate for therapy of severe infectious diseases. Omadacycline inhibits bacterial protein biosynthesis and competes with tetracycline for binding to the ribosome. Its interactions with the 70S ribosome were, therefore, analyzed in great detail and compared with tigecycline and tetracycline. All three antibiotics are inhibited by mutations in the 16S rRNA that mediate resistance to tetracycline in Brachyspira hyodysenteriae, Helicobacter pylori, Mycoplasma hominis, and Propionibacterium acnes. Chemical probing with dimethyl sulfate and Fenton cleavage with iron(II)-complexes of the tetracycline derivatives revealed that each antibiotic interacts in an idiosyncratic manner with the ribosome. X-ray crystallography had previously revealed one primary binding site for tetracycline on the ribosome and up to five secondary sites. All tetracyclines analyzed here interact with the primary site and tetracycline also with two secondary sites. In addition, each derivative displays a unique set of non-specific interactions with the 16S rRNA.

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