Abstract
Many bacterial species regulate the production of queuosine modified tRNAs by employing riboswitches selective for 7‐aminomethyl‐7‐deazaguanine (preQ1), an essential precursor in the biosynthesis of queuosine. Analogous to other riboswitch systems, the preQ1 riboswitch mediates a feedback mechanism between a target mRNA and a recognized small molecule metabolite. For class‐I preQ1 riboswitches this regulatory mechanism is initiated by the specific binding of preQ1 to an aptamer domain in the 5′‐untranslated of the mRNA, driving the formation of an alternative RNA fold, and leading to a down regulation of protein production at the transcriptional level. The molecular details of this binding event have been well described for the Bacillus subtilis riboswitch aptamer, but the current model fails to explain the high binding affinities observed for several preQ1 analogs, particularly those introducing carbonyl functional groups at the 7‐aminomethyl position. To investigate the origin of these favorable metabolite/RNA contacts we have designed a series of carbonyl containing analogs and have adapted a rapid fluorescence‐based binding assay to measure their affinities as a function of magnesium concentration. Binding data for these analogs will be presented for the Bacillus subtilis aptamer and several pathogenic sequences of variable loop‐I length, including Staphylococcus aureus. These results directly test a binding model requiring inner sphere magnesium coordination between the carbonyl oxygen of these analogs and a phosphodiester oxygen in loop‐I.Support or Funding InformationThis work was supported by the Murdock Trust, the National Aeronautics and Space Administration, the College of Science and Engineering of Seattle University and the Department of Bioengineering of the University of Washington.
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