Biology subjects

Batey, R. T.

Publications and source records attributed to Batey, R. T..

3 recordsLinked to original sources

bioRxiv2018

Structure-activity relationship of flavin analogs that target the FMN riboswitch

The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative --5FDQD-- that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC=\"FIGDIR/small/389148_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (41K):\norg.highwire.dtl.DTLVardef@f59798org.highwire.dtl.DTLVardef@1b38b02org.highwire.dtl.DTLVardef@6b50faorg.highwire.dtl.DTLVardef@1915fc4_HPS_FORMAT_FIGEXP M_FIG Exploring the chemical structure landscape of FMN riboswitch binders.\n\nC_FIG

biochemistry↗

bioRxiv2018

Requirements for efficient cotranscriptional regulatory switching in designed variants of the Bacillus subtilis pbuE adenine-responsive riboswitch

Riboswitches, generally located in the 5-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch informing the transcriptional or translational machinery. While the structure and function of riboswitch effector-binding (aptamer) domains have been intensely studied, only recently have the requirements for efficient linkage between small molecule binding and the structural switch in the cellular and cotranscriptional context begun to be actively explored. To address this, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches containing a secondary structural switch. Using a cell-based fluorescent protein reporter assay to assess ligand-dependent regulatory activity in E. coli, these studies revealed previously unrecognized features of the riboswitch. Most importantly, it was found that local and long-range conformational dynamics in two regions of the aptamer domain have a significant effect upon efficient regulatory switching. Further, sequence features of the expression platform including the pre-aptamer leader sequence, a nucleation helix and a putative programmed pause have clear affects upon ligand-dependent regulation. Together, these data point to sequence and structural features distributed throughout the riboswitch required to strike a balance between rates of ligand binding, transcription and secondary structural switching via a strand exchange mechanism.

biochemistry↗

bioRxiv2017

Development of a riboswitch-based platform for live cell imaging of RNAs in mammalian cells

RNAs directly regulate a vast array of critical cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular Riboglow platform leverages different color fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescent turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding U RNA in live mammalian cells. A direct side-by-side comparison revealed that Riboglow outperformed the dye binding aptamer Broccoli and performed on par with the current gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR/small/199240_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@11b5e3borg.highwire.dtl.DTLVardef@1c78fc0org.highwire.dtl.DTLVardef@aeba74org.highwire.dtl.DTLVardef@1531e3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗