Insights into the mechanism of a G-quadruplex-unwinding DEAH-box helicase.

The unwinding of nucleic acid secondary structures within cells is crucial to maintain genomic integrity and prevent abortive transcription and translation initiation. DHX36, also known as RHAU or G4R1, is a DEAH-box ATP-dependent helicase highly specific for DNA and RNA G-quadruplexes (G4s). A fundamental mechanistic understanding of the interaction between helicases and their G4 substrates is important to elucidate G4 biology and pave the way toward G4-targeted therapies. Here we analyze how the thermodynamic stability of G4 substrates affects binding and unwinding by DHX36. We modulated the stability of the G4 substrates by varying the sequence and the number of G-tetrads and by using small, G4-stabilizing molecules. We found an inverse correlation between the thermodynamic stability of the G4 substrates and rates of unwinding by DHX36. In stark contrast, the ATPase activity of the helicase was largely independent of substrate stability pointing toward a decoupling mechanism akin to what has been observed for many double-stranded DEAD-box RNA helicases. Our study provides the first evidence that DHX36 uses a local, non-processive mechanism to unwind G4 substrates, reminiscent of that of eukaryotic initiation factor 4A (eIF4A) on double-stranded substrates.Cancer Research UK and ERC (Balasubramanian group); Cambridge Trust studentship (to M.C.C.); Intramural Program of the National Heart, Lung and Blood Institute, NIH; ALS on the Berkeley Center for Structural Biology beamlines, US National Institutes of Health (NIH); NIH Oxford Cambridge Scholars Program [to M.C.C.]. Funding for open access charge: University of Cambridge.This is the final published version. The article was originally published in Nucleic Acids Research, 2015, Vol. 43, No. 4 2223–2231, doi: 10.1093/nar/gkv051

Validating the fragment-based drug discovery strategy for targeting biological RNAs: Lead fragments specifically bind and remodel the TPP riboswitch

Thiamine pyrophosphate (TPP) riboswitches regulate essential genes in bacteria by changing conformation upon binding intracellular TPP. Previous studies using fragment-based approaches identified small molecule “fragments” that bind this gene-regulatory mRNA domain. Crystallographic studies now show that, despite having micromolar Kds, four different fragments bind the TPP riboswitch site-specifically, occupying the pocket that recognizes the aminopyrimidine of TPP. Unexpectedly, the unoccupied site that would recognize the pyrophosphate of TPP rearranges into a structure distinct from that of the cognate complex. This idiosyncratic fragment-induced conformation, also characterized by small-angle X-ray scattering (SAXS) and chemical probing (SHAPE), represents a possible mechanism for adventitious ligand discrimination by the riboswitch, and suggests that off-pathway conformations of RNAs can be targeted for drug development. Our structures, together with previous screening studies, demonstrate the feasibility of fragment-based drug discovery against RNA targets

Cocrystal structure of a class-I preQ1 riboswitch reveals a pseudoknot recognizing an essential hypermodified nucleobase

Riboswitches are mRNA domains that bind metabolites and modulate gene expression in cis. We report cocrystal structures of a remarkably compact riboswitch (34 nucleotides suffice for ligand recognition) from Bacillus subtilis selective for the essential nucleobase preQ1 (7-aminomethyl-7-deazaguanine). These reveal a previously unrecognized pseudoknot fold, and suggest a conserved gene-regulatory mechanism whereby ligand binding promotes sequestration of an RNA segment that otherwise assembles into a transcriptional anti-terminator

Small self-cleaving ribozymes.

The hammerhead, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes catalyze sequence-specific intramolecular cleavage of RNA. They range between 50 and 150 nucleotides in length, and are known as the "small self-cleaving ribozymes." Except for the glmS ribozyme that functions as a riboswitch in Gram-positive bacteria, they were originally discovered as domains of satellite RNAs. However, recent studies show that several of them are broadly distributed in genomes of organisms from many phyla. Each of these ribozymes has a unique overall architecture and active site organization. Crystal structures have revealed how RNA active sites can bind preferentially to the transition state of a reaction, whereas mechanistic studies have shown that nucleobases can efficiently perform general acid-base and electrostatic catalysis. This versatility explains the abundance of ribozymes in contemporary organisms and also supports a role for catalytic RNAs early in evolution