Publicação

Molecular interactions within an antagonistic module of protein involved in plant developmental processes

Detalhes bibliográficos
Resumo:	The protein-protein interactions (PPIs) underlying the interplay between plant transcription factors (TFs) have not been studied in detail; however, understanding their interaction is important to unravel Gene Regulatory Networks. The Antirrhinum majus asymmetric flower development involves the proteins DIVARICATA (DIV), RADIALIAS (RAD) and DIV-and-RAD interacting factor (DRIF) working in the tug-of-war DIV-DRIF-RAD (DDR) regulatory module. It functions as an on/off molecular switch, in which DIV and DRIF can interact in a DNA-binding complex; RAD can sequester the DRIF in the cytoplasm, preventing the establishment of the DIV-DRIF complex. The module has been co-opted into different functions across several species. In Arabidopsis thaliana it is unclear whether the DDR homologous proteins work as a module, despite previous work showing involvement of individual genes in the control of several molecular processes. One of the aims of this work was to characterize the expression of the DDR genes in tissues and conditions in A. thaliana to unveil a possible involvement of the DDR regulatory module during development, as well as in the response to abiotic stresses and in the regulation of hormone signalling. Another aim of the work was to obtain recombinant protein to in the future perform biophysical assays to further characterize the molecular mechanism through which RAD antagonizes the DIV function by establishing a complex with the DRIF protein. Only AmRAD1 was previously expressed and purified. In this work, the conditions for the expression of soluble AmDIV1, MpDIV1, and AtDRIF3 were optimized. The last aim of the work was to determine the amino acid residues that establish PPI between AmRAD and AmDRIF binding through in silico analysis of the AmRAD1 crystal structure and the AmDRIF1 MYB simulated structure. Some AmRAD1 residues potentially involved in PPI were tested through site-directed mutagenesis and by yeast two-hybrid assays. The identified residues were utilized to restrict the simulated models of interaction between the AmRAD1 and AmDRIF1 structures. The results from this work will take us closer to understanding the DDR module by identifying new processes in which it is involved. It will also take us closer to understanding the RAD-DRIF and DIV-DRIF complexes, which may be used to fine tune the module into a system for graded expression, which would allow the regulation of the phenotype associated with the module.
Autores principais:	Sousa, Ângela Simone Gonçalves de
Assunto:	DIV-DRIF-RAD regulatory module Heterologous protein expression Protein-protein interactions Relative gene expression Expressão de proteínas heterólogas Expressão genética relativa Interações proteína-proteína Módulo regulatório DIV-DRIF-RAD
Ano:	2022
País:	Portugal
Tipo de documento:	dissertação de mestrado
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade do Minho
Idioma:	inglês
Origem:	RepositóriUM - Universidade do Minho