Reflections on apparent DNA bending by charge variants of bZIP proteins.
Basic-leucine zipper (bZIP) proteins have been studied intensely as transcription factors. It has been proposed that the bZIP domain might modulate transcription activation through the induction of conformational changes in the DNA binding site. We have been interested in using bZIP peptides as convenient models with which to study the role of asymmetric phosphate neutralization in DNA bending. DNA bending experiments have yielded discordant results for bZIP peptides studied by electrophoretic- vs solution-based assays. We review the history of DNA bending assays involving bZIP peptides and introduce the reader to examples of discordant results. Our recent published experiments designed to clarify this field of study will then be reviewed. The engineering of protein fusions has established that electrophoretic phasing assays are relatively insensitive to precise protein structure/conformation and instead appear to report DNA bending, as influenced by protein charge. New applications of time-resolved fluorescence resonance energy transfer (FRET) have allowed for the first time corroboration of electrophoretic phasing assays with solution-based FRET measurements. We report that two conventional DNA bending assays that rely on DNA ligation cannot be applied to analysis of the bZIP peptides we studied due to ligation inhibition.
Hardwidge
Philip R
PR
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905, USA.
Parkhurst
Kay M
KM
Parkhurst
Lawrence J
LJ
Maher
L James
LJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D051790:G-Box Binding Factors; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D006657:Histones; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA