Elucidation of the substrate of tRNA-modifying enzymes MnmEG leads to in vitro reconstitution of an evolutionarily conserved uridine hypermodification.

The evolutionarily conserved bacterial proteins MnmE and MnmG collectively install a carboxymethylaminomethyl (cmnm) group at the fifth position of wobble uridines of several tRNA species. While the reaction catalyzed by MnmEG is one of the central steps in the biosynthesis of the methylaminomethyl (mnm) posttranscriptional tRNA modification, details of the reaction remain elusive. Glycine is known to be the source of the carboxy methylamino moiety of cmnm, and a tetrahydrofolate (THF) analog is thought to supply the one carbon that is appended to the fifth position of U. However, the nature of the folate analog remains unknown. This article reports the in vitro biochemical reconstitution of the MnmEG reaction. Using isotopically labeled methyl and methylene THF analogs, we demonstrate that methylene THF is the true substrate. We also show that reduced FAD is required for the reaction and that DTT can replace the NADH in its role as a reductant. We discuss the implications of these methylene-THF and reductant requirements on the mechanism of this key tRNA modification catalyzed by MnmEG.

Insights into the Mechanism of Installation of 5-Carboxymethylaminomethyl Uridine Hypermodification by tRNA-Modifying Enzymes MnmE and MnmG.

The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm5) or a 5-taurinomethyl (τm5) group onto wobble uridines of several tRNA species. The Escherichia coli MnmE binds guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CH2THF), while MnmG binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH). Together with glycine, MnmEG catalyzes the installation of cmnm5 in a reaction that also requires hydrolysis of GTP. In this letter, we investigated key steps of the MnmEG reaction using a combination of biochemical techniques. We show multiple lines of evidence supporting flavin-iminium FADH[N5═CH2]+ as a central intermediate in the MnmEG reaction. Using a synthetic FADH[N5═CD2]+ analogue, the intermediacy of the FAD in the transfer of the methylene group from CH2THF to the C5 position of U34 was unambiguously demonstrated. Further, MnmEG reactions containing the deuterated flavin-iminium intermediate and alternate nucleophiles such as taurine and ammonia also led to the formation of the anticipated U34-modified tRNAs, showing FAD[N5═CH2]+ as the universal intermediate for all MnmEG homologues. Additionally, an RNA-protein complex stable to urea-denaturing polyacrylamide gel electrophoresis was identified. Studies involving a series of nuclease (RNase T1) and protease (trypsin) digestions along with reverse transcription experiments suggest that the complex may be noncovalent. While the conserved MnmG cysteine C47 and C277 mutant variants were shown to reduce FAD, they were unable to promote the modified tRNA formation. Overall, this study provides critical insights into the biochemical mechanism underlying tRNA modification by the MnmEG.

Synthesis of N2-Deoxyguanosine Modified DNAs and the Studies on Their Translesion Synthesis by the E. coli DNA Polymerase IV.

We report the synthesis of N2-aryl (benzyl, naphthyl, anthracenyl, and pyrenyl)-deoxyguanosine (dG) modified phosphoramidite building blocks and the corresponding damaged DNAs. Primer extension studies using E. coli Pol IV, a translesion polymerase, demonstrate that translesion synthesis (TLS) across these N2-dG adducts is error free. However, the efficiency of TLS activity decreases with increase in the steric bulkiness of the adducts. Molecular dynamics simulations of damaged DNA-Pol IV complexes reveal the van der Waals interactions between key amino acid residues (Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76, and Tyr79) of the enzyme and adduct that help to accommodate the bulky damages in a hydrophobic pocket to facilitate TLS. Overall, the results presented here provide insights into the TLS across N2-aryl-dG damaged DNAs by Pol IV.

Benzothiazole hydrazones of furylbenzamides preferentially stabilize c-MYC and c-KIT1 promoter G-quadruplex DNAs.

The stabilization of G-quadruplex DNA structures by using small molecule ligands having simple structural scaffolds has the potential to be harnessed for developing next generation anticancer agents. Because of the structural diversity of G-quadruplexes, it is challenging to design stabilizing ligands, which can specifically bind to a particular quadruplex topology. To address this, herein, we report the design and synthesis of three benzothiazole hydrazones of furylbenzamides having different side chains (ligands 1, 2 and 3), which show preferential stabilization of promoter quadruplex DNAs (c-MYC and c-KIT1) having parallel topologies over telomeric and duplex DNAs. The CD melting study revealed that all the ligands, in particular ligand 2, exhibit higher stabilization toward parallel promoter quadruplexes (ΔTm = 10-15 °C) as compared to antiparallel promoter quadruplex (h-RAS1), telomeric quadruplex and duplex DNAs (ΔTm = 0-3 °C). FID assay and fluorimetric titration results also reveal the preferential binding of ligands toward c-MYC and c-KIT1 promoter quadruplex DNAs over telomeric and duplex DNAs. Validating these results further, Taq DNA polymerase stop assay showed IC50∼ 6.4 µM for ligand 2 with the c-MYC DNA template, whereas the same for the telomeric DNA template was found to be >200 µM. Molecular modeling and dynamics studies demonstrated a 1 : 1 binding stoichiometry in which stacking and electrostatic interactions play important roles in stabilizing the c-MYC G-quadruplex structure. Taken together, the results presented here provide new insights into the design of structurally simple scaffolds for the preferential stabilization of a particular G-quadruplex topology.

Direct Nanopore Sequencing for the 17 RNA Modification Types in 36 Locations in the E. coli Ribosome Enables Monitoring of Stress-Dependent Changes.

The bacterium Escherichia coli possesses 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Nanopore direct RNA sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to unmodified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knockout E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modification's signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. The double modification m4Cm1402 resides in 16S rRNA, and it decreased with both stressors. Using the helicase dwell time, it was determined that the N4 methyl group is lost during both stressors, and the 2'-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and the earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to all seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.

Systematic Mutation and Unnatural Base Pair Incorporation Improves Riboswitch-Based Biosensor Response Time.

Engineered RNAs have applications in diverse fields from biomedical to environmental. In many cases, the folding of the RNA is critical to its function. Here we describe a strategy to improve the response time of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal base pairs. Both natural and unnatural base pair mutants were found to improve the biosensor response time without compromising fold turn-on or ligand affinity. These strategies can be transferred to improve the performance of other RNA-based tools.

Site-Specific Profiling of 4-Thiouridine Across Transfer RNA Genes in Escherichia coli.

The transfer RNA (tRNA) modification 4-thiouridine (s4U) acts as a near-ultraviolet (UVA) radiation sensor in Escherichia coli (E. coli), where it induces a growth delay upon exposure to the UVA radiation (∼310-400 nm). Herein, we report sequencing methodology for site-specific profiling of s4U modification in E. coli tRNAs. Upon the addition of iodoacetamide (IA) or iodoacetyl-PEG2-biotin (BIA), the nucleophilic sulfur of s4U forms a reaction product that is extensively characterized by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. This method is readily applied to the alkylation of natively occurring s4U on E. coli tRNA. Next-generation sequencing of BIA-treated tRNA from E. coli revealed misincorporations at position 8 in 19 of the 20 amino acid tRNA species. Alternatively, tRNA from the ΔthiI strain, which cannot introduce the s4U modification, does not exhibit any misincorporation at the corresponding positions, directly linking the base transitions and the tRNA modification. Independently, the s4U modification on E. coli tRNA was further validated by LC-MS/MS sequencing. Nuclease digestion of wild-type and deletion strains E. coli tRNA with RNase T1 generated smaller s4U/U containing fragments that could be analyzed by MS/MS analysis for modification assignment. Furthermore, RNase T1 digestion of tRNAs treated either with IA or BIA showed the specificity of iodoacetamide reagents toward s4U in the context of complex tRNA modifications. Overall, these results demonstrate the utility of the alkylation of s4U in the site-specific profiling of the modified base in native cellular tRNA.