Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding

G-quadruplexes (G4s) are unusual DNA structures and can stall DNA replication, causing genomic instability for the cell. Although the solved crystal structure of the DHX36 helicase demonstrated that G4 was specifically targeted by a DHX36-specific motif (DSM), lack of complete structural details for general G4-resolving helicases without specific target motifs remains a barrier to the complete understanding of the molecular basis underlying the recognition and unfolding of G4s. Herein, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif) complexed with a G4, thereby mimicking the physiological G4 formed during DNA replication. Strictly different from the previous determined G4-helicase structure of DHX36, our structure revealed that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B constituting a G4-Recognizing Surface (GRS). The overall topology of the G4 structure solved in this work maintains its three-layered propeller-type G4 topology, with no significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 were differentially recognized by GRS residues mainly through electrostatic, ionic interactions and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Our structure explains how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.

Prebiotic Pathway from Ribose to RNA Formation

At the focus of abiotic chemical reactions is the synthesis of ribose. No satisfactory explanation was provided as to the missing link between the prebiotic synthesis of ribose and prebiotic RNA (preRNA). Hydrogen cyanide (HCN) is assumed to have been the principal precursor in the prebiotic formation of aldopentoses in the formose reaction and in the synthesis of ribose. Ribose as the best fitting aldopentose became the exclusive sugar component of RNA. The elevated yield of ribose synthesis at higher temperatures and its protection from decomposition could have driven the polymerization of the ribose-phosphate backbone and the coupling of nucleobases to the backbone. RNA could have come into being without the involvement of nucleotide precursors. The first nucleoside monophosphate is likely to have appeared upon the hydrolysis of preRNA contributed by the presence of reactive 2′-OH moieties in the preRNA chain. As a result of phosphorylation, nucleoside monophosphates became nucleoside triphosphates, substrates for the selective synthesis of genRNA.

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.

Global analysis of lysine acetylation reveals its role in multiple biological processes in Glycine max

Protein lysine acetylation (Kac) is an important post-translational modification present in both animal and plant cells. Here, we reported the results from a proteomic investigation of Kac in soybean leaves. We totally identified 3148 acetylation sites in 1538 proteins from three biological replicates, among 59 lysine acetylation sites in core histones, represents the largest acetylome dataset in plants to date. Gene Ontology (GO) functional analysis illustrated that most of the acetylated proteins involved in metabolic processes (include carboxylic acid metabolic process, oxoacid metabolic process, nucleoside metabolic process, nucleoside phosphate metabolic process, and ribose phosphate metabolic process). KEGG pathway enrichment showed Kac plays an important role in Photosynthesis, Carbon fixation in photosynthetic organisms and Citrate cycle (TCA cycle). Meanwhile we also find a total of 17 conserved Kac motifs. All together, our study not only provides the first global and most extensive lysine acetylation analysis in soybean leaves, but also suggest that lysine acetylation is play an important and unique role in plants.

Classification of RNA backbone conformations into rotamers using 13C′ chemical shifts: exploring how far we can go

The conformational space of the ribose-phosphate backbone is very complex as it is defined in terms of six torsional angles. To help delimit the RNA backbone conformational preferences, 46 rotamers have been defined in terms of these torsional angles. In the present work, we use the ribose experimental and theoretical 13C′ chemical shifts data and machine learning methods to classify RNA backbone conformations into rotamers and families of rotamers. We show to what extent the experimental 13C′ chemical shifts can be used to identify rotamers and discuss some problem with the theoretical computations of 13C′ chemical shifts.

Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak

The mechanisms of adaptation to inactivation of essential genes remain unknown. Here we inactivate E. coli dihydrofolate reductase (DHFR) by introducing D27G,N,F chromosomal mutations in a key catalytic residue with subsequent adaptation by an automated serial transfer protocol. The partial reversal G27- > C occurred in three evolutionary trajectories. Conversely, in one trajectory for D27G and in all trajectories for D27F,N strains adapted to grow at very low metabolic supplement (folAmix) concentrations but did not escape entirely from supplement auxotrophy. Major global shifts in metabolome and proteome occurred upon DHFR inactivation, which were partially reversed in adapted strains. Loss-of-function mutations in two genes, thyA and deoB, ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP. Multiple evolutionary pathways of adaptation converged to a suboptimal solution due to the high accessibility to loss-of-function mutations that block the path to the highest, yet least accessible, fitness peak.

A genetically adaptable strategy for ribose scavenging in a human gut symbiont plays a diet-dependent role in colon colonization

SummaryEfficient nutrient acquisition in the competitive human gut is essential for microbial persistence. While polysaccharides have been well-studied nutrients for the gut microbiome, other resources such as co-factors and nucleic acids have been less examined. We describe a series of ribose utilization systems (RUSs) that are broadly represented in Bacteroidetes and appear to have diversified to allow access to ribose from a variety of substrates. OneBacteroides thetaiotaomicronRUS variant is critical for competitive gut colonization in a diet-specific fashion. Using molecular genetics, we probed the nature of the ribose source underlying this diet-specific phenotype, revealing that hydrolytic functions in RUS (e.g., to cleave ribonucleosides) are present but dispensable. Instead, ribokinases that are activatedin vivoand participate in cellular ribose-phosphate metabolism are essential. Our results underscore the extensive mechanisms that gut symbionts have evolved to access nutrients and how metabolic context determines the impact of these functionsin vivo.