RESUMO
RNA polymerase (RNAP) is a major target of gene regulation. Thermus thermophilus bacteriophage P23-45 encodes two RNAP binding proteins, gp39 and gp76, which shut off host gene transcription while allowing orderly transcription of phage genes. We previously reported the structure of the T. thermophilus RNAPâ¢σA holoenzyme complexed with gp39. Here, we solved the structure of the RNAPâ¢σA holoenzyme bound with both gp39 and gp76, which revealed an unprecedented inhibition mechanism by gp76. The acidic protein gp76 binds within the RNAP cleft and occupies the path of the template DNA strand at positions -11 to -4, relative to the transcription start site at +1. Thus, gp76 obstructs the formation of an open promoter complex and prevents transcription by T. thermophilus RNAP from most host promoters. gp76 is less inhibitory for phage transcription, as tighter RNAP interaction with the phage promoters allows the template DNA to compete with gp76 for the common binding site. gp76 also inhibits Escherichia coli RNAP highlighting the template-DNA binding site as a new target site for developing antibacterial agents.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Thermus thermophilus/enzimologia , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófagos/genética , Bacteriófagos/fisiologia , Sítios de Ligação , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Interações Hospedeiro-Patógeno , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Domínios Proteicos , Thermus thermophilus/genética , Thermus thermophilus/virologia , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Linear and miktoarm star-shaped diblock copolymers consisting of single-stranded DNA and poly(N-isopropylacrylamide) (PNIPAAm) with various compositions were synthesized via atom transfer radical polymerization and click chemistry. The temperature-responsive phase transition behavior, micellization, was systematically examined using UV-vis spectrometry, high-sensitivity differential scanning calorimetry, dynamic light scattering, and small-angle X-ray scattering. The lower critical solution temperature (LCST) increased, and its enthalpy decreased with decreasing PNIPAAm content. The copolymers self-assembled into well-defined nanoparticles having a core composed of PNIPAAm and a coronal layer of DNA above LCST. The particle size and micellar aggregation number of copolymer chains depended on the macromolecular composition and chain architecture. On the other hand, regardless of their factors, the surface area occupied by one DNA strand was found to be almost unchanged. The hybridization of DNA on the nanoparticles with fully complementary one induced the aggregation of the particles in a non-cross-linking configuration. The nanoparticle composed of miktoarm star copolymer showed a quicker DNA-hybridization response in this non-cross-linking aggregation compared with the case of a linear analogue.
Assuntos
Resinas Acrílicas/química , DNA de Cadeia Simples/química , Micelas , Temperatura , Sequência de Bases , Coloides , DNA de Cadeia Simples/genética , Nanopartículas/química , Hibridização de Ácido Nucleico , Transição de FaseRESUMO
Poly(N-isopropylacrylamide) (PNIPAAm) grafted with single-stranded (ss) DNA conjugate (PNIPAAm-g-DNA) self-assembles above its lower critical solution temperature to form colloidal particles. When the ssDNA within the particle hybridizes with its complementary DNA, the particles aggregate above a certain threshold of salt concentration with drastically increased turbidity in solution. Detailed structural information of the particle was obtained mainly by small-angle X-ray scattering. The influence of copolymer composition on the morphology of particle and non-crosslinking aggregation was examined. The particle consists of hydrophobic PNIPAAm core surrounded by hydrophilic DNA strands. The increase in DNA fraction brought about a significant decrease in core size, whereas the shell thickness little changed and corresponded to the length of DNA. A structural model with a sticky potential was applied to the analysis of particle aggregate. This analysis provided that the particles aggregate while the coronal layers interpenetrate each other. The interaction between the particles was quantified in terms of the sticky potential and showed a trend to be influenced by the particle size rather than the graft density of DNA strands on the particle.
