RESUMO
Stimuli-responsive switching molecules have been widely investigated for the purpose of the mechanical control of biomolecules. Recently developed arylazopyrazole (AAP) shows photoisomerization activity, displaying a faster response to light-induced conformational changes and unique absorption spectral properties compared with those of conventionally used azobenzene. Herein, it is demonstrated that AAP can be used as a photoswitching molecule to control photoinduced assembly and disassembly of DNA origami nanostructures. An AAP-modified DNA origami has been designed and constructed. It is observed that the repeated assembly and disassembly of AAP-modified X-shaped DNA origami and hexagonal origami with complementary strands can be achieved by alternating UV and visible-light irradiation. Closed and linear assemblies of AAP-modified X-shaped origami were successfully formed by photoirradiation, and more than 1â µm linear assemblies were formed. Finally, it is shown that the two photoswitches, AAP and azobenzene, can be used in tandem to independently control different assembly configurations by using different irradiation wavelengths. AAP can extend the variety of available wavelengths of photoswitches and stably result in the assembly and disassembly of various DNA origami nanostructures.
Assuntos
DNA/química , DNA/efeitos da radiação , Luz , Nanoestruturas/química , Nanoestruturas/efeitos da radiação , Conformação de Ácido Nucleico/efeitos da radiação , Processos Fotoquímicos/efeitos da radiaçãoRESUMO
Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand 'azoCm' was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.
Assuntos
Aptâmeros de Nucleotídeos/química , Compostos Azo/química , Conformação de Ácido Nucleico/efeitos da radiação , RNA/química , Aptâmeros de Nucleotídeos/efeitos da radiação , Fenômenos Biofísicos , Ligantes , Luz , RNA/efeitos da radiação , Riboswitch/efeitos da radiação , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The interplay between multiple chromophores in nucleic acids and photosynthetic proteins gives rise to complex electronic phenomena and largely governs the de-excitation dynamics. Electronic coupling between bases in the excited states of single strands of DNA and RNA may extend over several bases and likely protects nucleic acids from harmful UV damage. Here we report on the coupling between bases in single RNA strands of cytosine and find that the excited state is delocalized over up to five bases at neutral pH, where all bases are non-protonated (i.e. neutral). Delocalization is over four bases at 278 nm excitation, while it involves five bases at shorter wavelengths of 188 nm and 201 nm. This is in contrast to only nearest-neighbour interactions for corresponding DNA strands as previously reported. The current results seemingly corroborate earlier findings of larger spatial communication in RNA than in DNA strands of adenine, but there is no obvious link between the overall structure of strands and delocalization lengths. RNA cytosine strands form a tight helix, while comparatively, adenine strands show less tight packing, also compared to their DNA counterparts, and yet exhibit even higher delocalisation.
Assuntos
Citosina/química , Citosina/efeitos da radiação , RNA/química , RNA/efeitos da radiação , Fenômenos Eletromagnéticos , Conformação de Ácido Nucleico/efeitos da radiação , Raios UltravioletaRESUMO
The ordered structure of UV chromophores in DNA resembles photosynthetic light-harvesting complexes in which quantum coherence effects play a major role in highly efficient directional energy transfer. The possible role of coherent excitons in energy transport in DNA remains debated. Meanwhile, energy transport properties are greatly important for understanding the mechanisms of photochemical reactions in cellular DNA and for DNA-based artificial nanostructures. Here, we studied energy transfer in DNA complexes formed with silver nanoclusters and with intercalating dye (acridine orange). Steady-state fluorescence measurements with two DNA templates (15-mer DNA duplex and calf thymus DNA) showed that excitation energy can be transferred to the clusters from 21 and 28 nucleobases, respectively. This differed from the DNA-acridine orange complex for which energy transfer took place from four neighboring bases only. Fluorescence up-conversion measurements showed that the energy transfer took place within 100 fs. The efficient energy transport in the Ag-DNA complexes suggests an excitonic mechanism for the transfer, such that the excitation is delocalized over at least four and seven stacked bases, respectively, in one strand of the duplexes stabilizing the clusters. This result demonstrates that the exciton delocalization length in some DNA structures may not be limited to just two bases.
Assuntos
DNA/química , Transferência de Energia/efeitos da radiação , Conformação de Ácido Nucleico/efeitos da radiação , Laranja de Acridina/química , Animais , Bovinos , DNA/genética , DNA/efeitos da radiação , Fluorescência , Nanoestruturas/química , Fotossíntese/genética , Fotossíntese/efeitos da radiação , Teoria Quântica , Prata/química , Raios UltravioletaRESUMO
The prokaryotic RNA chaperone Hfq mediates sRNA-mRNA interactions and plays a significant role in post-transcriptional regulation of the type III secretion (T3S) system produced by a range of Escherichia coli pathotypes. UV-crosslinking was used to map Hfq-binding under conditions that promote T3S and multiple interactions were identified within polycistronic transcripts produced from the locus of enterocyte effacement (LEE) that encodes the T3S system. The majority of Hfq binding was within the LEE5 and LEE4 operons, the latter encoding the translocon apparatus (SepL-EspADB) that is positively regulated by the RNA binding protein, CsrA. Using the identified Hfq-binding sites and a series of sRNA deletions, the sRNA Spot42 was shown to directly repress translation of LEE4 at the sepL 5' UTR. In silico and in vivo analyses of the sepL mRNA secondary structure combined with expression studies of truncates indicated that the unbound sepL mRNA is translationally inactive. Based on expression studies with site-directed mutants, an OFF-ON-OFF toggle model is proposed that results in transient translation of SepL and EspA filament assembly. Under this model, the nascent mRNA is translationally off, before being activated by CsrA, and then repressed by Hfq and Spot42.
Assuntos
Translocação Bacteriana/genética , Proteínas de Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Sítios de Ligação/genética , Citoesqueleto/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Conformação de Ácido Nucleico/efeitos da radiação , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/efeitos da radiação , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/efeitos da radiação , Raios UltravioletaRESUMO
We developed an efficient, versatile, and accessible super-resolution microscopy method to construct a nanoparticle assembly at a spatial resolution below the optical diffraction limit. The method utilizes DNA and a photoactivated DNA cross-linker. Super-resolution optical techniques have been used only as a means to make measurements below the light diffraction limit. Furthermore, no optical technique is currently available to construct nanoparticle assemblies with a precisely designed shape and internal structure at a resolution of a few tens of nanometers (nm). Here we demonstrate that we can fulfill this deficiency by utilizing spontaneous structural dynamics of DNA hairpins combined with single-molecule fluorescence resonance energy transfer (smFRET) microscopy and a photoactivated DNA cross-linker. The stochastic fluorescence blinking due to the spontaneous folding and unfolding motions of DNA hairpins enables us to precisely localize a folded hairpin and solidify it only when it is within a predesigned target area whose size is below the diffraction limit. As the method is based on an optical microscope and an easily clickable DNA cross-linking reagent, it will provide an efficient means to create large nanoparticle assemblies with a shape and internal structure at an optical super-resolution, opening a wide window of opportunities toward investigating their photophysical and optoelectronic properties and developing novel devices.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Microscopia de Fluorescência , Nanopartículas/química , DNA/efeitos da radiação , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Luz , Conformação de Ácido Nucleico/efeitos da radiação , Estimulação Luminosa , Imagem Individual de MoléculaRESUMO
In this study, an accurately and digitally regulated allosteric nanoswitch based on the conformational control of two DNA hairpins was developed. By switching between UV irradiation and blue light conditions, the second molecular beacon (H#2) would bind/separate with a repression sequence (RES) via the introduced PTG molecules (a photosensitive azobenzene derivative), resulting in the target aptamer sequence in the first molecular beacon (H#1) not being able/being able to hold the stem-loop configuration, hence losing/regaining the ability to bind with the target. Importantly, we successfully monitor conformation changes of the nanoswitch by an elegant mathematical model for connecting Ki (the dissociation constant between RES and H#2) with Kd (the overall equilibrium constant of the nanoswitch binding the target), hence realizing "observing" DNA structure across dimensions from "structural visualization" to digitization and, accurately, digitally regulating DNA structure from digitization to "structural visualization".
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Nanoestruturas/química , Compostos Azo/química , Compostos Azo/efeitos da radiação , DNA/metabolismo , DNA/efeitos da radiação , Sequências Repetidas Invertidas , Ligantes , Modelos Químicos , Conformação de Ácido Nucleico/efeitos da radiação , Trombina/metabolismo , Raios UltravioletaRESUMO
UV light can induce chemical reactions in nucleic acids and thereby damage the genetic code. Like all of the five primary nucleobases, the isolated RNA base uracil exhibits ultrafast, nonradiative relaxation after photoexcitation, which helps to avoid photodamage most of the time. Nevertheless, within RNA and DNA strands, commonly occurring photolesions have been reported and are often ascribed to long-lived and delocalized excited states. Our quantum dynamical study now shows that excited-state longevity can also occur on a single nucleobase, without the need for delocalization. We include the effects of an atomistic RNA surrounding in wave packet simulations and explore the photorelaxation of uracil in its native biological environment. This reveals that steric hindrance through embedding in an RNA strand can inhibit the ultrafast relaxation mechanism of uracil, promoting excited-state longevity and potential photodamage. This process is nearly independent from the specific combination of neighboring bases.
Assuntos
RNA/química , Uracila/química , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos da radiação , Fotólise/efeitos da radiação , Teoria Quântica , Raios UltravioletaRESUMO
Structure-specific helicases, such as RecG, play an important role in the resolution of recombination intermediates. A bioinformatic analysis of mycobacterial genomes led to the identification of a protein (RecGwed ) with a C-terminal "edge" domain, similar to the wedge domain of RecG. RecGwed is predominately found in the phylum Actinobacteria and in few human pathogens. Mycobacterium smegmatis RecGwed was able to bind branched DNA structures in vitro but failed to interact with single- or double-stranded DNA. The expression of recGwed in M. smegmatis cells was up-regulated during stationary phase/UV damage and down-regulated during MMS/H2 O2 treatment. These observations indicate the possible involvement of RecGwed in transactions during recombination events, that proceed though branched DNA intermediates. © 2018 IUBMB Life, 70(8):786-794, 2018.
Assuntos
Biologia Computacional , DNA Helicases/genética , Genoma Bacteriano/genética , Mycobacterium smegmatis/genética , DNA/efeitos dos fármacos , DNA/genética , DNA/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , DNA Helicases/química , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Mycobacterium smegmatis/enzimologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos da radiação , Raios UltravioletaRESUMO
Manually controlling siRNA activity is an essentially important way to spatiotemporally investigate gene expression and function. Owing to ease of operation and precise manipulation, light can be used for controlled regulation of siRNA-induced gene silencing. Here, we developed a series of caged siRNAs with folic acid modification at the 5' terminus of the antisense strand of the siRNA through a photolabile linker. The attachment of the folic acid moiety temporarily masked the corresponding siRNA activity. Upon illumination, these caged siRNAs were activated, and their gene silencing activities were restored. Based on this strategy, we successfully photomodulated gene expression of both an exogenous gene (for green fluorescent protein, GFP) and an endogenous gene (for mototic kinesin-5, Eg5) in cells.
Assuntos
Ácido Fólico/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Sequência de Bases , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Cinesinas/genética , Luz , Conformação de Ácido Nucleico/efeitos da radiação , Processos Fotoquímicos , Interferência de RNA/efeitos da radiação , RNA Interferente Pequeno/farmacocinéticaRESUMO
Ligand binding influences the dynamics of the DNA helix in both the binding site and adjacent regions. This, in particular, is reflected in the changing pattern of cleavage of complexes under the action of ultrasound. The specificity of ultrasound-induced cleavage of the DNA sugar-phosphate backbone was studied in actinomycin D (AMD) complexes with double-stranded DNA restriction fragments. After antibiotic binding, the cleavage intensity of phosphodiester bonds between bases was shown to decrease at the chromophore intercalation site and to increase in adjacent positions. The character of cleavage depended on the sequences flanking the binding site and the presence of other AMD molecules bound in the close vicinity. A comparison of ultrasonic and DNase I cleavage patterns of AMD-DNA complexes provided more detail on the local conformation and dynamics of the DNA double helix in both binding site and adjacent regions. The results pave the way for developing a novel approach to studies of the nucleotide sequence dependence of DNA conformational dynamics and new techniques to identify functional genome regions.
Assuntos
Sequência de Bases/genética , Pegada de DNA/métodos , DNA/genética , Expressão Gênica/efeitos da radiação , Sítios de Ligação , DNA/química , DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dactinomicina/química , Desoxirribonuclease I/química , Expressão Gênica/genética , Substâncias Intercalantes/química , Ligantes , Conformação de Ácido Nucleico/efeitos da radiação , Ondas UltrassônicasRESUMO
Self-folding origami is of great interest in current research on functional materials and structures, but there is still a challenge to develop a simple method to create freestanding, reversible, and complex origami structures. This communication provides a feasible solution to this challenge by developing a method based on the digit light processing technique and desolvation-induced self-folding. In this new method, flat polymer sheets can be cured by a light field from a commercial projector with varying intensity, and the self-folding process is triggered by desolvation in water. Folded origami structures can be recovered once immersed in the swelling medium. The self-folding process is investigated both experimentally and theoretically. Diverse 3D origami shapes are demonstrated. This method can be used for responsive actuators and the fabrication of 3D electronic devices.
Assuntos
Fotoquímica , Polímeros/química , Luz , Nanotecnologia , Conformação de Ácido Nucleico/efeitos da radiação , Polímeros/efeitos da radiação , Água/químicaRESUMO
Light-harvesting complexes collect light energy and deliver it by a cascade of energy and electron transfer processes to the reaction center where charge separation leads to storage as chemical energy. The design of artificial light-harvesting assemblies faces enormous challenges because several antenna chromophores need to be kept in close proximity but self-quenching needs to be avoided. Double stranded DNA as a supramolecular scaffold plays a promising role due to its characteristic structural properties. Automated DNA synthesis allows incorporation of artificial chromophore-modified building blocks, and sequence design allows precise control of the distances and orientations between the chromophores. The helical twist between the chromophores, which is induced by the DNA framework, controls energy and electron transfer and thereby reduces the self-quenching that is typically observed in chromophore aggregates. This Account summarizes covalently multichromophore-modified DNA and describes how such multichromophore arrays were achieved by Watson-Crick-specific and DNA-templated self-assembly. The covalent DNA systems were prepared by incorporation of chromophores as DNA base substitutions (either as C-nucleosides or with acyclic linkers as substitutes for the 2'-deoxyribofuranoside) and as DNA base modifications. Studies with DNA base substitutions revealed that distances but more importantly relative orientations of the chromophores govern the energy transfer efficiencies and thereby the light-harvesting properties. With DNA base substitutions, duplex stabilization was faced and could be overcome, for instance, by zipper-like placement of the chromophores in both strands. For both principal structural approaches, DNA-based light-harvesting antenna could be realized. The major disadvantages, however, for covalent multichromophore DNA conjugates are the poor yields of synthesis and the solubility issues for oligonucleotides with more than 5-10 chromophore modifications in a row. A logical alternative approach is to leave out the phosphodiester bridges between the chromophores and let chromophore-nucleoside conjugates self-assemble specifically along single stranded DNA as template. The self-organization of chromophores along the DNA template based on canonical base pairing would be advantageous because sequence selective base pairing could provide a structural basis for programmed complexity within the chromophore assembly. The self-assembly is governed by two interactions. The chromophore-nucleoside conjugates as guest molecules are recognized via hydrogen bonds to the corresponding counter bases in the single stranded DNA template. Moreover, the π-π interactions between the stacked chromophores stabilize these self-assembled constructs with increasing length. Longer DNA templates are more attractive for self-assembled antenna. The helicity in the stack of porphyrins as guest molecules assembled on the DNA template can be switched by environmental changes, such as pH variations. DNA-templated stacks of ethynyl pyrene and nile red exhibit left-handed chirality, which stands in contrast to similar covalent multichromophore-DNA conjugates with enforced right-handed helicity. With ethynyl nile red, it is possible to occupy every available binding site on the templates. Mixed assemblies of ethynyl pyrene and nile red show energy transfer and thereby provide a proof-of-principle that simple light-harvesting antennae can be obtained in a noncovalent and self-assembled fashion. With respect to the next important step, chemical storage of the absorbed light energy, future research has to focus on the coupling of sophisticated DNA-based light-harvesting antenna to reaction centers.
Assuntos
DNA/química , Biomimética , Luz , Conformação de Ácido Nucleico/efeitos da radiação , Processos FotoquímicosRESUMO
Ultraviolet (UV) radiation is a leading external hazard to the integrity of DNA. Exposure to UV radiation triggers a cascade of chemical reactions, and many molecular products (photolesions) have been isolated that are potentially dangerous for the cellular system. The early steps that take place after UV absorption by DNA have been studied by ultrafast spectroscopy. The review focuses on the evolution of excited electronic states, the formation of photolesions, and processes suppressing their formation. Emphasis is placed on lesions involving two thymine bases, such as the cyclobutane pyrimidine dimer, the (6-4) lesion, and its Dewar valence isomer.
Assuntos
Dano ao DNA/efeitos da radiação , DNA/genética , Animais , DNA/química , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos da radiação , Processos Fotoquímicos , Raios UltravioletaRESUMO
In order to obtain the energy deposition pattern of ionizing radiation in the nanometric scale of genetic material and to investigate the different sensitivities of the DNA conformations, direct effects of (60)Co gamma rays on the three A, B and Z conformations of DNA have been studied. For this purpose, single-strand breaks (SSB), double-strand breaks (DSB), base damage (BD), hit probabilities and three microdosimetry quantities (imparted energy, mean chord length and lineal energy) in the mentioned DNA conformations have been calculated and compared by using GEometry ANd Tracking 4 (Geant4) toolkit. The results show that A-, B- and Z-DNA conformations have the highest yields of DSB (1.2 Gy(-1) Gbp(-1)), SSB (25.2 Gy(-1) Gbp(-1)) and BD (4.81 Gy(-1) Gbp(-1)), respectively. Based on the investigation of direct effects of radiation, it can be concluded that the DSB yield is largely correlated to the topological characteristics of DNA models, although the SSB yield is not. Moreover, according to the comparative results of the present study, a reliable candidate parameter for describing the relationship between DNA damage yields and geometry of DNA models in the theoretical radiation biology research studies would be the mean chord length (4 V/S) of the models.
Assuntos
Radioisótopos de Cobalto/efeitos adversos , Dano ao DNA , DNA de Forma B/química , DNA Forma Z/química , Raios gama/efeitos adversos , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos da radiação , DNA de Forma B/genética , DNA Forma Z/genética , Relação Dose-Resposta à Radiação , RadiometriaRESUMO
Two-photon-regulated, shape-changing DNA nanostructures are demonstrated by integrating a DNA nanotube with a two-photon photocleavable module that enables the opening of the cavities of tube, and becomes partially single-stranded in response to two-photon excitation under 800 nm fs laser pulses.
Assuntos
DNA/química , Nanotubos/efeitos da radiação , Fótons , Polimerização , DNA/efeitos da radiação , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Nanotubos/química , Conformação de Ácido Nucleico/efeitos da radiação , Polimerização/efeitos da radiação , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Photoinduced processes in nucleic acids are phenomena of fundamental interest in diverse fields, from prebiotic studies, through medical research on carcinogenesis, to the development of bioorganic photodevices. In this contribution we survey many aspects of the research across the boundaries. Starting from a historical background, where the main milestones are identified, we review the main findings of the physical-chemical research of photoinduced processes on several types of nucleic-acid fragments, from monomers to duplexes. We also discuss a number of different issues which are still under debate.
Assuntos
Ácidos Nucleicos/efeitos da radiação , Purinas/efeitos da radiação , Pirimidinas/efeitos da radiação , Raios Ultravioleta , Pareamento de Bases/efeitos da radiação , Conformação de Ácido Nucleico/efeitos da radiação , Ácidos Nucleicos/química , Processos Fotoquímicos , Purinas/química , Pirimidinas/químicaRESUMO
Four novel photoactivated binitroimidazole prodrugs were synthesized. These agents produced DNA interstrand cross-links (ICLs) and direct strand breaks (DSB) upon UV irradiation, whereas no or very few DNA ICLs and DSBs were observed without UV treatment. Although these four molecules (1-4) contain the same binitroimidazole moiety, they bear four different leaving groups, which resulted in their producing different yields of DNA damage. Compound 4, with nitrogen mustard as a leaving group, showed the highest ICL yield. Surprisingly, compounds 1-3, without any alkylating functional group, also induced DNA ICL formation, although they did so with lower yields, which suggested that the binitroimidazole moiety released from UV irradiation of 1-3 is capable of cross-linking DNA. The DNA cross-linked products induced by these compounds were completely destroyed upon 1.0 M piperidine treatment at 90 °C (leading to cleavage at dG sites), which revealed that DNA cross-linking mainly occurred via alkylation of dGs. We proposed a possible mechanism by which alkylating agents were released from these compounds. HRMS and NMR analysis confirmed that free nitrogen mustards were generated by UV irradiation of 4. Suppression of DNA ICL and DSB formation by a radical trap, TEMPO, indicated the involvement of free radicals in the photo reactions of 3 and 4 with DNA. On the basis of these data, we propose that UV irradiation of compounds 1-4 generated a binitroimidazole intermediate that cross-links DNA. The higher ICL yield observed with 4 resulted from the amine effector nitrogen mustard released from UV irradiation.
Assuntos
Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Quebras de DNA/efeitos dos fármacos , Quebras de DNA/efeitos da radiação , DNA/genética , Nitroimidazóis/química , Nitroimidazóis/farmacologia , Alquilantes/química , Alquilantes/farmacologia , DNA/química , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos da radiação , Raios UltravioletaRESUMO
The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG-3' human telomere sequence can fold into G-quadruplexes that adopt the hybrid, basket, or propeller fold. The telomere sequence is hypersensitive to UV-induced thymine dimer (T=T) formation, yet it does not cause telomere shortening. In this work, the potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand why this damage is tolerated in telomeres. First, established methods, such as thermal melting measurements, electrophoretic mobility shift assays, and circular dichroism spectroscopy, were utilized to determine the effects of the damage on these structures. Second, a single-molecule ion channel recording technique using α-hemolysin (α-HL) was employed to examine further the structural differences between the damaged sequences. It was observed that the damage caused slightly lower thermal stabilities and subtle changes in the circular dichroism spectra for hybrid and basket folds. The α-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are tolerated in edgewise and diagonal loops. The largest change was observed for the T=T-containing natural telomere sequence when the propeller fold (all double-chain reversal loops) was studied. On the basis of the α-HL experiments, it was determined that a triplexlike structure exists under conditions that favor a propeller structure. The biological significance of these observations is discussed.
Assuntos
Quadruplex G , Dímeros de Pirimidina/química , Telômero/química , Telômero/genética , Toxinas Bacterianas , Sequência de Bases , Dicroísmo Circular , Dano ao DNA , Ensaio de Desvio de Mobilidade Eletroforética , Quadruplex G/efeitos da radiação , Proteínas Hemolisinas , Humanos , Modelos Moleculares , Nanoporos , Conformação de Ácido Nucleico/efeitos da radiação , Telômero/efeitos da radiação , Raios UltravioletaRESUMO
DnaK-DnaJ-GrpE and GroES-GroEL are the major chaperone machineries in bacteria. In many species, dnaKJ and groESL are encoded in bicistronic operons. Quantitative proteomics revealed that DnaK and GroEL amounts in Salmonella dominate over DnaJ and GroES respectively. An imperfect transcriptional terminator in the intergenic region of dnaKJ is known to result in higher transcript levels of the first gene. Here, we examined the groESL operon and asked how the second gene in a heat shock operon can be preferentially expressed and found that an RNA structure in the 5'untranslated region of groES is responsible. The secondary structure masks the Shine-Dalgarno (SD) sequence and AUG start codon and thereby modulates translation of groES mRNA. Reporter gene assays combined with structure probing and toeprinting analysis revealed a dynamic temperature-sensitive RNA structure. Following an increase in temperature, only the second of two RNA hairpins melts and partially liberates the SD sequence, thus facilitating translation. Translation of groEL is not temperature-regulated leading to an excess of the chaperonin in the cell at low temperature. Discussion in a broader context shows how structured RNA segments can differentially control expression of temperature-affected operons in various ways.