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

Monitoring and Scoring Counter-Diffusion Protein Crystallization Experiments in Capillaries by in situ Dynamic Light Scattering

In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D

A MITF Mutation Associated with a Dominant White Phenotype and Bilateral Deafness in German Fleckvieh Cattle

A dominantly inherited syndrome associated with hypopigmentation, heterochromia irides, colobomatous eyes and bilateral hearing loss has been ascertained in Fleckvieh cattle (German White Fleckvieh syndrome). This syndrome has been mapped to bovine chromosome (BTA) 22 using a genome-wide association study with the bovine high density single nucleotide polymorphism array. An R210I missense mutation has been identified within microphthalmia-associated transcription factor (MITF) as responsible for this syndrome. The mutation is located in the highly conserved basic region of the protein and causes a negative-dominant effect. SOX10 and PAX3 promoter binding site mutations in MITF could be ruled out as causative for the German White Fleckvieh syndrome. Molecular characterization of this newly detected bovine syndrome means a large animal model is now available for the Tietz syndrome in humans

Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Pseudouridine (Ψ) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Ψ55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Ψ55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein

A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination

RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab–ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs′ high molecular weight, large surface area and phasing power.National Institutes of Health (U.S.) (GM61835

Cleavage mediated by the P15 domain of bacterial RNase P RNA

Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3′-end of tRNA precursors resulting in the formation of the RCCA–RNase P RNA interaction (interacting residues underlined) in the bacterial RPR–substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg2+ at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for ‘RNA-mediated’ cleavage of RNA

A Selective Na+ Aptamer Dissected by Sensitized Tb3+ Luminescence

This is the peer reviewed version of the following article: Zhou, W., Ding, J., & Liu, J. (2016). A Selective Na+ Aptamer Dissected by Sensitized Tb3+ Luminescence. Chembiochem, 17(16), 1563–1570. https://doi.org/10.1002/cbic.201600174, which has been published in final form at https://doi.org/10.1002/cbic.201600174. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.A previous study of two RNA-cleaving DNAzymes, NaA43 and Ce13d, revealed the possibility of a common Na+ aptamer motif. Because Na+ binding to DNA is a fundamental biochemical problem, the interaction between Ce13d and Na+ was studied in detail by using sensitized Tb3+ luminescence spectroscopy. Na+ displaces Tb3+ from the DNAzyme, and thus quenches the emission from Tb3+. The overall requirement for Na+ binding includes the hairpin and the highly conserved 16-nucleotide loop in the enzyme strand, along with a few unpaired nucleotides in the substrate. Mutation studies indicate good correlation between Na+ binding and cleavage activity, thus suggesting a critical role of Na+ binding for the enzyme activity. Ce13d displayed a K-d of approximate to 20mm with Na+ (other monovalent cations: 40-60mm). The K-d values for other metal ions are mainly due to non-specific competition. With a single nucleotide mutation, the specific Na+ binding was lost. Another mutant improved K-d to 8mm with Na+. This study has demonstrated a Na+ aptamer with important biological implications and analytical applications. It has also defined the structural requirements for Na+ binding and produced an improved mutant.University of Waterloo; Natural Sciences and Engineering Research Council of Canada (NSERC); Foundation for Shenghua Scholar of Central South University; National Natural Science Foundation of China [21301195]; China Scholarship Council (CSC) [201406370116