The relationship between intranuclear mobility of the NF-kappaB subunit p65 and its DNA binding affinity.
It has been hypothesized that the main determinant of the intranuclear mobility of transcription factors is their ability to bind DNA. In the present study, we have extensively tested the relationship between the intranuclear mobility of the NF-kappaB subunit p65 and binding to its consensus target sequence. The affinity of p65 for this binding site is altered by mutation of specific acetylation sites, so these mutants provide a model system to study the relationship between specific DNA binding affinity and intranuclear mobility. DNA binding affinity was measured in vitro using an enzyme-linked immunosorbent assay-based method, and intranuclear mobility was measured using the fluorescence recovery after photobleaching technique on yellow fluorescent protein-tagged p65 constructs. A negative correlation was observed between DNA binding affinity and intranuclear mobility of p65 acetylation site mutants. However, moving the yellow fluorescent protein tag from the C terminus of p65 to the N terminus resulted in an increased mobility but did not significantly affect DNA binding affinity. Thus, all changes in DNA binding affinity produce alterations in mobility, but not vice versa. Finally, a positive correlation was observed between mobility and the randomness of the intranuclear distribution of p65. Our data are in line with a model in which the intranuclear mobility and distribution of a transcription factor are determined by its affinity for specific DNA sequences, which may be altered by protein-protein interactions.
Schaaf
Marcel J M
MJM
Department of Asthma Biology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY. Electronic address: schaaf@rulbim.leidenuniv.nl.
Willetts
Lynsey
L
Department of Asthma Biology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY; School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.
Hayes
Brian P
BP
Department of Asthma Biology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY.
Maschera
Barbara
B
Department of Asthma Biology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY.
Stylianou
Eleni
E
School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.
Farrow
Stuart N
SN
Department of Asthma Biology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY.
D000107:Acetylation; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D006801:Humans; D016297:Mutagenesis, Site-Directed; D038761:Photobleaching; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport; D051996:Transcription Factor RelA / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D051996:Transcription Factor RelA; D004247:DNA