RESUMO
Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is 'nicked' rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I-DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I's binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs.
Assuntos
DNA/genética , Desoxirribonuclease I/genética , Endonucleases/genética , Complexos Multiproteicos/genética , Bacillus/enzimologia , DNA/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Desoxirribonuclease I/ultraestrutura , Endonucleases/ultraestrutura , Cinética , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido NucleicoRESUMO
The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved ß-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.
Assuntos
Grânulos Citoplasmáticos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Sítios de Ligação , Proliferação de Células , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/patologia , Feminino , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.
Assuntos
Nucleoproteínas/química , Proteínas de Ligação a RNA/ultraestrutura , Proteína 1 de Ligação a Y-Box/ultraestrutura , Sequência de Aminoácidos/genética , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Escherichia coli/genética , Humanos , Nucleoproteínas/genética , Nucleoproteínas/ultraestrutura , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Dobramento de Proteína , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribossomos/química , Ribossomos/genética , Proteína 1 de Ligação a Y-Box/química , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
á : A hallmark of neurodegenerative diseases is the accumulation of cytoplasmic protein aggregates in neurons of affected subjects. Among recently identified elements of these aggregates are RNA-binding proteins (RBPs) involved in RNA metabolism and alternative splicing and have in common the presence of low complexity domains (LCD) that are prone to self-assemble and form aggregates. The mechanism of cytoplasmic protein aggregation remains elusive. Stress granules (SGs) that are micrometric RNA-protein assemblies located in the cytoplasm of cells exposed to environmental stress are suspected to play the role of seeds. The review sheds light on the recent experimental results that suggest a link between SGs and cytoplasmic protein aggregates but also propose other routes for the formation of these aggregates. PURPOSE OF REVIEW: To analyze the potential relationship between cytoplasmic protein aggregates in neurons of affected subjects and stress granules. RECENT FINDINGS: Liquid phase separation explains how protein and RNA could assemble in membraneless compartments, notably SGs. These results highlight the importance of RBPs with LCD in the SG assembly. Maturation of SGs and in particular the dense core is a potential source of insoluble protein aggregates. Several lines of evidence linked stress granule dynamics to pathogenic protein aggregates. (i) Proteins that accumulate in cytoplasmic aggregates are also SG components. (ii) Neurons are specifically exposed to stress events due to their high metabolism and long lifespan. (iii) Diseases linked protein mutations affect the SG dynamics. (iv) SG dense core could be a breeding ground for protein aggregates. However, we should also keep in mind that SGs are not the only RNA-protein assembly in the cytoplasm; the RNA transport granules could also play a role in the formation of insoluble protein aggregates.
