RESUMEN
Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF-DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: 'associated' (73° of DNA bend), 'half-wrapped' (107°) and 'fully-wrapped' (147°), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.
Asunto(s)
ADN Bacteriano/genética , Factores de Integración del Huésped/genética , Conformación de Ácido Nucleico , Nucleoproteínas/genética , Empaquetamiento del ADN/genética , ADN Bacteriano/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Escherichia coli/genética , Factores de Integración del Huésped/ultraestructura , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Nucleoproteínas/ultraestructura , Imagen Individual de MoléculaRESUMEN
In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the X-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (~300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA.
Asunto(s)
Proteínas de Unión al ADN/ultraestructura , Factores de Integración del Huésped/ultraestructura , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Sitios de Unión/genética , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Interacciones Huésped-Patógeno/genética , Humanos , Factores de Integración del Huésped/genética , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Mycobacterium tuberculosis/patogenicidad , Conformación Proteica en Hélice alfa/genética , Streptomyces coelicolor/genética , Tuberculosis/genéticaRESUMEN
Bacterial nucleoid is a dynamic entity that changes its three-dimensional shape and compaction depending on cellular physiology. While these changes are tightly associated with compositional alterations of abundant nucleoid-associated proteins implicated in reshaping the nucleoid, their cooperation in regular long-range DNA organization is poorly understood. In this study, we reconstitute a novel nucleoprotein structure in vitro, which is stabilized by cooperative effects of major bacterial DNA architectural proteins. While, individually, these proteins stabilize alternative DNA architectures consistent with either plectonemic or toroidal coiling of DNA, the combination of histone-like protein, histone-like nucleoid structuring protein, and integration host factor produces a conspicuous semiperiodic structure. By employing a bottom-up in vitro approach, we thus characterize a minimum set of bacterial proteins cooperating in organizing a regular DNA structure. Visualized structures suggest a mechanism for nucleation of topological transitions underlying the reshaping of DNA by bacterial nucleoid-associated proteins.
