Imaging and mapping protein-binding sites on DNA regulatory regions with atomic force microscopy.
Regulation of gene expression is fundamental in biological systems. A systematic search for protein binding sites in gene promoters has been done in recent years. Biochemical techniques are easy and reliable when analysing protein interactions with short pieces of DNA, but are difficult and tedious when long pieces of DNA have to be analysed. Here we propose AFM as a reliable and easy technique for identifying protein interaction sites in long DNA molecules like gene promoters. We support this idea using a well-known model: the interaction of the Pho4 protein with the PHO5 gene promoter. We have also applied the technique to demonstrate that Mig1 protein binds to two motifs in the promoter of HXK2 gene. Our results allow us to define Mig1p as a new factor probably contributing to the carbon source-dependent transcription regulation of HXK2 gene.
Moreno-Herrero
F
F
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, 33006 Oviedo, Spain.
Herrero
P
P
Colchero
J
J
Baró
A M
AM
Moreno
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli; D005656:Fungal Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics*; D018625:Microscopy, Atomic Force / Q000379:methods; D028061:Phosphate Transport Proteins; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C085976:MIG1 protein, S cerevisiae; D026901:Membrane Transport Proteins; C060292:PHO4 protein, S cerevisiae; D028061:Phosphate Transport Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C055831:phosphate permease