Showing papers on "Base pair published in 2016"
TL;DR: PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells, is developed and provides novel insights into the RNA structurome and interactome.
492 citations
TL;DR: This work presents a general solution to this problem that offers the ability for nonspecialists to design and synthesize nearly arbitrary DNA-based nanoparticles using only a simple surface-based representation of target 3D geometry to automatically generate the ssDNA needed to synthesize the object.
Abstract: Scaffolded DNA origami is a versatile means of synthesizing complex molecular architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, we report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asymmetric polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biological as well as nonbiological applications.
466 citations
01 Jan 2016
TL;DR: In this paper, the authors constructed hybrid genes between Escherichia coli lacZ gene and the iso-l-cytochrome c (CYC1) gene of Saccharomyces cerevistae by recombination in vitro.
Abstract: Hybrid genes between the Escherichia coli lacZ gene and the iso-l-cytochrome c (CYC1) gene of Saccharomyces cerevistae were constructed by recombination in vitro. Each of the hybrid genes encodes a chimeric protein with a cytochrome c moiety at the amino terminus and an active f8-galactosidase (fi-D- galactoside galactohydrolase, EC 3.2.1.23) moiety at the carboxy terminus. When these hybrids are introduced into S. cerevisiae on plasmid vectors, they direct synthesis of B3-galactosidase. /3- Galactosidase levels directed by one such plasmid display the pat- tern of regulation normally seen for cytochrome c (i.e., a reduction of synthesis in cells grown in glucose). This plasmid.contains one codon of CYCi fused to lacZ, and the fused gene is preceded by the 1100 nucleotides that lie upstream from CYC1. An analysis of deletions in the upstream DNA suggests that sequences required for efficient transcription initiation of CYC1 lie within the DNA segment 250-700 base pairs upstream from the start of the CYC1 coding sequence. This region is at least 130 base pairs upstream from the "Hogness box" sequence that precedes the CYC1 coding
323 citations
01 May 2016
TL;DR: In this article, a top-down strategy was proposed to design nearly arbitrary DNA architectures autonomously based only on target shape, represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes.
Abstract: Scaffolded DNA origami is a versatile means of synthesizing complex molecular architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, we report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asymmetric polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biological as well as nonbiological applications.
294 citations
TL;DR: LIGR-seq data reveal unexpected interactions between small nucleolar (sno)RNAs and m RNAs, including those involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs.
287 citations
01 Jan 2016
TL;DR: In this paper, specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl transferase, EC 2.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before the RNA initialization site.
Abstract: Specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl- transferase, EC 2.7.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before the RNA initia- tion site. Two of these contain nucleotide sequences that are fairly conserved among many promoters, known as the "Prib- now box" and "-35 region" homologies; the third, just upstream from the Pribnow box, is not conserved. The polymerase binds preferentially to the coding strand and for the most part touches only one face of the DNA helix.
267 citations
TL;DR: Single-molecule FRET is used to probe real-time interactions between Cas9–RNA and DNA targets and observes at least two different bound FRET states that may represent distinct steps in target search and proofreading.
Abstract: Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from 2 s(-1) upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s(-1)), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.
215 citations
TL;DR: It is proposed that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phageDNA, but not the host DNA.
209 citations
TL;DR: The 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation is presented, suggesting a plausible mechanism of remodelling before exon ligation.
Abstract: Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 A cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.
200 citations
TL;DR: The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre, including striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces.
Abstract: U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 A resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.
185 citations
TL;DR: Structural comparison between the AsPf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf 1)-crRNA binary complex identifies a unique mechanism employed by CpF1 for target recognition.
Abstract: CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRISPR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an "open-to-closed" conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9.
TL;DR: The data provides a quantitative basis for the rational design of dynamic DNA-based nanoscale machines and assemblies and may inform kinetic models of DNA hybridization and may help in adjusting force fields to perform more realistic molecular dynamics simulations.
Abstract: INTRODUCTION In DNA double helices, hydrogen bonds connect the base pairs across the two strands, and stacking bonds act along the helical axis between neighboring base pairs. Our understanding of DNA and the way it is processed in biology would profit from improved knowledge about the elementary bonds in DNA. Detailed knowledge of the time scales for breaking and forming individual base pairs and base-pair stacks would also help to make more informed decisions in the design of dynamic DNA-based nanoscale devices. RATIONALE The goal of this work is to measure the dynamics of DNA base-pair stacking at the level of individual base-pair steps. Because stacking interactions act perpendicularly to the hydrogen bonds, it should be possible to use mechanical forces to break stacking while leaving hydrogen bonds intact. To realize such measurements, we combine the positioning capabilities of DNA origami with single-molecule manipulation, as enabled by dual-beam optical traps. To make the weak single–base-pair stacking interactions experimentally accessible, we prepared parallel arrays of blunt-end DNA double helices to take advantage of avidity effects when these arrays form stacking interactions (see the figure). Our design allowed controlling the number and the sequences of the base-pair stacks. Noise-suppressing by stiff DNA origami beams connected by a flexible polymer tether enabled the repeated detection of unbinding and rebinding of stacking contacts at low forces (down to 2 piconewtons) with high time resolution (up to 1 kHz). RESULTS We sampled all 16 sequence combinations of installing a particular interfacial base pair on the array on the left beam and another base pair in the array on the right beam, and we created arrays with two, four, and six blunt ends. We could measure the force-dependent lifetimes for all base-pair step sequence combinations in the presence of 20 mM MgCl 2 , which is a condition typically used in DNA nanotechnology. For a subset of base-pair step combinations, we also obtained data in the presence of 500 mM NaCl, which mimics the conditions in the cell nucleus. The base-pair stack arrays spontaneously dissociated at average rates ranging from 0.02 to 500 per second, where the dissociation time scale strongly depended on the sequence combination and the stack array size. For a given sequence combination, larger array sizes always had larger lifetimes, as expected from avidity. Another key feature revealed in the lifetime data was the low sensitivity of the stacking interactions on the extent of pulling force. This phenomenon reflects short-ranged interaction potentials. Concerning rebinding of the stack arrays, we found that the rebinding kinetics depended much more strongly on the applied force, which may be understood by considering that rebinding of the stacks requires a thermally activated contraction of the flexible tether—which was the same for all variants—against an opposing force tether contraction. However, the rebinding kinetics was independent, within experimental error, of the base-pair step sequence combination and the size of the array under study. We used a model to estimate the free-energy increments per single base-pair stack from the kinetic rates that we measured with stack arrays. The free-energy increments per stack ranged from –0.8 to –3.4 kilocalories per mole. Our data reveals a trend in the stacking-strength hierarchy that may be associated with the extent of geometrical atomic overlap between the bases within a base-pair step. CONCLUSION Our data provides a quantitative basis for the rational design of dynamic DNA-based nanoscale machines and assemblies. Nanoengineers can directly read off the expected lifetimes of stack arrays for all sequence combinations and for various array sizes and at salt conditions that are commonly used in the field. With this data, design solutions for transition kinetics may be generated that cover several orders of magnitudes in lifetime, from milliseconds to several seconds. The sequence-resolved information obtained in our experiments may inform kinetic models of DNA hybridization and may help in adjusting force fields to perform more realistic molecular dynamics simulations. More generally, our experimental methods advance the capabilities of single-molecule mechanical experiments. Using the tethered-beam system, target molecules may be placed and exposed in controlled orientations and stoichiometry so as to study the weak forces occurring between them in solution. A variety of interactions between various kinds of molecules may be studied in the future due to the modularity and the addressability of the DNA origami–based tethered-beam system.
TL;DR: The 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target is presented, providing the structural basis for understanding the PAM-dependent directional R-loop formation process.
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 A crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 A apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.
TL;DR: The observed imino proton nuclear magnetic resonance resonances and Förster resonance energy transfer efficiencies suggest that m(6)A selectively destabilizes the portion of the hairpin stem where the U5-tract is located, increasing the solvent accessibility of the neighboring bases while maintaining the overall hairpin structure.
TL;DR: This review highlights the contribution Fourier transform infrared (FTIR) spectroscopy has made to the understanding of DNA conformation in relation to hydration and its potential role in clinical diagnostics and the importance of preserving the B-DNA conformation for the diagnosis of cancer.
Abstract: Since Watson and Crick's historical papers on the structure and function of DNA based on Rosalind Franklin's and Maurice Wilkin's X-ray diffraction patterns tremendous scientific curiosity has been aroused by the unique and dynamic structure of the molecule of life. A-DNA and B-DNA represent different conformations of the DNA molecule, which is stabilised by hydrogen interactions between base pairs, stacking interactions between neighboring bases and long-range intra- and inter-backbone forces. This review highlights the contribution Fourier transform infrared (FTIR) spectroscopy has made to the understanding of DNA conformation in relation to hydration and its potential role in clinical diagnostics. The review will first begin by elucidating the main forms of DNA conformation found in nature and the general structures of the A, B and Z forms. This is followed by a detailed critique on infrared spectroscopy applied to DNA conformation highlighting pivotal studies on isolated DNA, polynucleotides, nucleoprotein and nucleohistone complexes. A discussion on the potential of diagnosing cancer using FTIR spectroscopy based on the detection of DNA bands in cells and tissues will ensue, highlighting the recent studies investigating the conformation of DNA in hydrated and dehydrated cells. The method of hydration as a way to facilitate DNA conformational band assignment will be discussed and the conformational change to the A-form upon dehydration will be used to explain the reason for the apparent lack of FTIR DNA signals observed in fixed or air-dried cells and tissues. The advantages of investigating B-DNA in the hydrated state, as opposed to A-DNA in the dehydrated state, are exemplified in a series of studies that show: (1) improved quantification of DNA in cells; (2) improved discrimination and reproducibility of FTIR spectra recorded of cells progressing through the cell cycle; (3) insights into the biological significance of A-DNA as evidenced by an interesting study on bacteria, which can survive desiccation and at the same time undergo the B–A–B transition. Finally, the importance of preserving the B-DNA conformation for the diagnosis of cancer is put forward as way to improve the sensitivity of this powerful technique.
TL;DR: It is shown that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro and to explain the elusive role of CRISPR leader sequences during spacer Acquisition.
TL;DR: This evolution of DNA CT chemistry is described, from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology.
TL;DR: The progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms are surveyed, and the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules are considered.
Abstract: There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA-ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules.
TL;DR: It is concluded that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ.
TL;DR: Interestingly, aspercryptin is formed from compounds produced by two separate gene clusters, one of which makes the well-known product cichorine, raising the exciting possibility that fungi use differential regulation of expression of secondary metabolite gene clusters to increase the diversity of metabolites they produce.
Abstract: To reduce the secondary metabolite background in Aspergillus nidulans and minimize the rediscovery of compounds and pathway intermediates, we created a "genetic dereplication" strain in which we deleted eight of the most highly expressed secondary metabolite gene clusters (more than 244,000 base pairs deleted in total). This strain allowed us to discover a novel compound that we designate aspercryptin and to propose a biosynthetic pathway for the compound. Interestingly, aspercryptin is formed from compounds produced by two separate gene clusters, one of which makes the well-known product cichorine. This raises the exciting possibility that fungi use differential regulation of expression of secondary metabolite gene clusters to increase the diversity of metabolites they produce.
TL;DR: This study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials.
Abstract: The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA 'alphabet' by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces, reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid (PNA) all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials.
01 Jan 2016
TL;DR: In this paper, the authors studied the DNA replication problem in Drosophila melanogaster by electron microscopic examination of the DNA from rapidly dividing cleavage nuclei and found that the fine structure of the forks is very similar to that observed for the replication forks of bi-directionally replicating bacteriophage DNAs.
Abstract: The replicating chromosomlall DNA in Drosophila melanogaster cleavage nuclei has been visual- ized in the electron microscope as a serial array of closely spaced replicated regions created by pairs of diverging replication forks. The fine structure of the forks is very similar to that observed for the replication forks of bi- directionally replicating bacteriophage DNAs. However, the mean length of the single-stranded gaps in Drosophila forks is less than 200 nucleotide residues, much shorter than the gaps in phage forks. This difference in gap length corresponds to the observed difference in the size of Okazaki fragments from Drosophila and phage. The pleasing concept that the genetic information in a eukaryotic chromosome is contained in a single rmolecule of double-stranded DNA is supported by recent experiments with Drosophila (1) and yeast (2, 3). Given such a molecular continuity, the problem of reproducing the genetic order in a chromosome is reduced to the problem of replicating a single long DNA molecule which, for the largest chromosome in the fruit fly, Drosophila melanogaster, has a length of' about 2.1 cm., or 62,000 kb (ref. 1; kb (kilo bases) is a unit of length equal to 1000 bases or base pairs in single-stranded or double- stranded nucleic acids). We have studied this replication problem in D. melanogaster by electron microscopic examination of the DNA from rapidly dividing cleavage nuclei. At 24°, the cleavage nuclei divide every 9.6 min and exhibit an interphase of only 3.4 miii (4),
TL;DR: It is shown that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields, indicating that nucleotide formation and selection may have been robust processes on the prebiotics Earth, if other nucleobases preceded those of extant life.
Abstract: The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life.
TL;DR: Control functions of nucleoside modifications in gene regulation with a focus on recent findings on protein synthesis control by tRNA base modifications are summarized.
Abstract: Both, DNA and RNA nucleoside modifications contribute to the complex multi-level regulation of gene expression. Modified bases in tRNAs modulate protein translation rates in a highly dynamic manner. Synonymous codons, which differ by the third nucleoside in the triplet but code for the same amino acid, may be utilized at different rates according to codon–anticodon affinity. Nucleoside modifications in the tRNA anticodon loop can favor the interaction with selected codons by stabilizing specific base pairs. Similarly, weakening of base pairing can discriminate against binding to near-cognate codons. mRNAs enriched in favored codons are translated in higher rates constituting a fine-tuning mechanism for protein synthesis. This so-called codon bias establishes a basic protein level, but sometimes it is necessary to further adjust the production rate of a particular protein to actual requirements, brought by, e.g., stages in circadian rhythms, cell cycle progression or exposure to stress. Such an adjustment is realized by the dynamic change of tRNA modifications resulting in the preferential translation of mRNAs coding for example for stress proteins to facilitate cell survival. Furthermore, tRNAs contribute in an entirely different way to another, less specific stress response consisting in modification-dependent tRNA cleavage that contributes to the general down-regulation of protein synthesis. In this review, we summarize control functions of nucleoside modifications in gene regulation with a focus on recent findings on protein synthesis control by tRNA base modifications.
TL;DR: These findings suggest that the ATP hydrolysis‐driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.
Abstract: ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here,Methanococcus jannaschii MR-ATPγS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.
TL;DR: It is shown that G-C+ and A-U Hoogsteen base pairs are strongly disfavored in A-RNA, providing a mechanism for disrupting RNA structure through post-transcriptional modifications.
Abstract: The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson-Crick or Hoogsteen configurations. Here, we show that G-C(+) (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N(1)-methyladenosine and N(1)-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C(+) and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.
TL;DR: The crystal structures of the ATPγS‐bound dimer of the Rad50NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11RBD (Rad50‐ binding domain) along with small‐angle X‐ray scattering and cross‐linking studies are reported.
Abstract: The Mre11–Rad50–Nbs1 (MRN) complex is a central factor in the repair of DNA double‐strand breaks (DSBs). The ATP‐dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology‐mediated end‐joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS‐bound dimer of the Rad50 NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum ( Ct ) in complex with either DNA or Ct Mre11 RBD (Rad50‐binding domain) along with small‐angle X‐ray scattering and cross‐linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo . Our analyses provide a structural framework for the architecture of the eukaryotic Mre11–Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP‐dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.
TL;DR: This article designs a kinetic pathway based on toehold-exchange mechanisms that enables rearrangement of DNA bonds without the need for thermal denaturation, and test it on suspensions of DNA-functionalized liposomes, demonstrating tunability of aggregation rates over more than 1 order of magnitude.
Abstract: The selectivity of Watson-Crick base pairing has allowed the design of DNA-based functional materials bearing an unprecedented level of accuracy. Examples include DNA origami, made of tiles assembling into arbitrarily complex shapes, and DNA coated particles featuring rich phase behaviors. Frequently, the realization of conceptual DNA-nanotechnology designs has been hampered by the lack of strategies for effectively controlling relaxations. In this article, we address the problem of kinetic control on DNA-mediated interactions between Brownian objects. We design a kinetic pathway based on toehold-exchange mechanisms that enables rearrangement of DNA bonds without the need for thermal denaturation, and test it on suspensions of DNA-functionalized liposomes, demonstrating tunability of aggregation rates over more than 1 order of magnitude. While the possibility to design complex phase behaviors using DNA as a glue is already well recognized, our results demonstrate control also over the kinetics of such systems.
TL;DR: A Markov State Model is built from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail, and reveals that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved.
Abstract: The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3'-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3'-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.
TL;DR: The origins, form, and function of RNA secondary structure are reviewed, focusing on plants, and an overview of methods for probing secondary structure is provided, from physical methods such as X-ray crystallography and nuclear magnetic resonance (NMR) imaging to chemical and nuclease probing methods.
Abstract: RNA transcripts fold into secondary structures via intricate patterns of base pairing. These secondary structures impart catalytic, ligand binding, and scaffolding functions to a wide array of RNAs, forming a critical node of biological regulation. Among their many functions, RNA structural elements modulate epigenetic marks, alter mRNA stability and translation, regulate alternative splicing, transduce signals, and scaffold large macromolecular complexes. Thus, the study of RNA secondary structure is critical to understanding the function and regulation of RNA transcripts. Here, we review the origins, form, and function of RNA secondary structure, focusing on plants. We then provide an overview of methods for probing secondary structure, from physical methods such as X-ray crystallography and nuclear magnetic resonance (NMR) imaging to chemical and nuclease probing methods. Combining these latter methods with high-throughput sequencing has enabled them to scale across whole transcriptomes, yielding tremendous new insights into the form and function of RNA secondary structure.