18 research outputs found
Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser.
Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5\u27-monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1â
Ă
resolution. This model, which was refined against a data set from âŒ750 diffraction images (each from a single crystal), was found to be consistent with that previously obtained from data collected at 100â
K using conventional synchrotron X-radiation. These experiments demonstrate the feasibility of time-resolved XFEL experiments to understand how the ZTP riboswitch accommodates cognate ligand binding
Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination
Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusBâNusEâBoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8ânt that are recognized by the NusBâNusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key proteinâprotein and proteinâRNA interactions. Further crystallographic investigation of a NusBâNusEâdsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex
Mix-and-Inject Serial Femtosecond Crystallography to Capture RNA Riboswitch Intermediates
Time-resolved structure determination of macromolecular conformations and ligand-bound intermediates is extremely challenging, particularly for RNA. With rapid technological advances in both microfluidic liquid injection and X-ray free electron lasers (XFEL), a new frontier has emerged in time-resolved crystallography whereby crystals can be mixed with ligand and then probed with X-rays (mix-and-inject) in real time and at room temperature. This chapter outlines the basic setup and procedures for mix-and-inject experiments for recording time-resolved crystallographic data of riboswitch RNA reaction states using serial femtosecond crystallography (SFX) and an XFEL
Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser
Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5âČ-monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1â
Ă
resolution. This model, which was refined against a data set from âŒ750 diffraction images (each from a single crystal), was found to be consistent with that previously obtained from data collected at 100â
K using conventional synchrotron X-radiation. These experiments demonstrate the feasibility of time-resolved XFEL experiments to understand how the ZTP riboswitch accommodates cognate ligand binding
Co-crystal structure of the i Mango-III fluorescent RNA aptamer using an X-ray free-electron laser
Turn-on aptamers are in vitro-selected RNAs that bind to conditionally fluorescent small molecules and enhance their fluorescence. Upon binding TO1-biotin, the iMango-III aptamer achieves the largest fluorescence enhancement reported for turn-on aptamers (over 5000-fold). This aptamer was generated by structure-guided engineering and functional reselection of the parental aptamer Mango-III. Structures of both Mango-III and iMango-III have previously been determined by conventional cryocrystallography using synchrotron X-radiation. Using an X-ray free-electron laser (XFEL), the room-temperature iMango-IIIâTO1-biotin co-crystal structure has now been determined at 3.0â
Ă
resolution. This structural model, which was refined against a data set of âŒ1300 diffraction images (each from a single crystal), is largely consistent with the structures determined from single-crystal data sets collected at 100â
K. This constitutes a technical benchmark on the way to XFEL pumpâprobe experiments on fluorescent RNAâsmall molecule complexes
RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting
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