Caracterización mediante Microscopía de Fuerzas Atómicas de complejos de regulación transcripcional

Resumen

This work presents the structural and functional characterization by Atomic Force Microscopy (AFM) of different nucleoprotein complexes involved in transcriptional regulation. Therefore its main objective is to study regulation mechanisms by resolving the implicated structures using a single molecule technique. The Introduction has two sections: first it describes the AFM technique and its application in Biology. Secondly it describes the mechanisms that regulate gene transcription, and more specifically those that involve ¿transcription factor¿ proteins. This section also reviews the contributions of AFM to the study of nucleoprotein complexes. The special requirements of the technique when applied to these samples are presented in the Results chapters. The training in the AFM technique took place along the development of a second project: the structural characterization of the macromolecular proteins known as ¿chaperonins¿. The results obtained for these systems are used in the first section of the Introduction to illustrate and discuss the characteristics, possibilities and limitations of the AFM. The Results section presents the four regulation systems studied in this thesis work. The results for each system are introduced and discussed in a separate subchapter. The AFM results are merged to pre-existing biochemical and biophysical data and the new information is used to propose functional models. Chapter IV.1 presents the regulation system of the promoter PTtgGHI, of Pseudomonas putida. The repressor TtgV binds to the promoter region distorting the structure of the linear DNA. The protein-DNA complex is characterized at the single molecule level with AFM and the angle induced in the DNA by the protein is estimated from a statistical analysis of the distribution found in the structures observed. Chapter IV.2 presents the regulation system of the promoter PTodX of Pseudomonas putida. DNA structures of the binary and ternary complexes with the transcription factors TodT and IHF are described. The role of TodT repressor and that of the activator IHF are discussed according to the observed structures. Chapter IV.3 presents the regulation mechanism of the promoter PN of Azoarcus sp. CIB. The superstructure found for the nucleoprotein complex formed by the repressor BzdR when binding to the region of the promoter is described. The large complexity of the regulation model and the high resolution structural information provided by AFM to the study of this kind of systems is discussed, complementing and enriching existing data provided by biochemical and biophysical techniques. Chapter IV.4 presents the system most extensively characterized in this thesis work: transcription regulation in the bacteriophage Phi29. This highly modulated process involves two transcriptional factors, p4 and p6, and the genome region containing promoters A2c, A2b and A3, located at the boundary between early and late transcribed genes. Building from the ideas presented earlier and using statistical approaches, this chapter presents and discusses the results acquired for the structure of complexes of growing complexity ([p4-DNA], [p6-DNA], [RNAp- DNA], [p4-p6-DNA] and [p4-p6-RNAp-DNA]). Control experiments follow and then a regulatory model suggested by these results is proposed. The transcription regulating complex of another virus, ¿Nf¿, was studied to explore if the functional structure suggested for the Phi29 system was shared by analogous viruses. The discussion section analyses the difficulties derived from the study of these complexes, including both sample preparation for AFM and analysis of the results. Furthermore, all the structures resolved are compared and analysed in relation to their function in their corresponding regulatory models. General features are concluded at the end of this chapter and listed in the Conclusions final section.