5-hydroxytryptophan as a new intrinsic probe for investigating protein-DNA interactions by analytical ultracentrifugation. Study of the effect of DNA on self-assembly of the bacteriophage lambda cI repressor.
Pairwise cooperativity between proteins bound to DNA is believed to be important in governing the transcriptional regulation of numerous genes. However, the spectral overlap of normal proteins and DNA has blocked the study of these interactions by many physical methods. As shown recently by Ross et al. (in press), lambda cI repressor spectrally enhanced by 5-hydroxytryptophan (5-OHTrp), expressed in vivo using an Escherichia coli tryptophan auxotroph, exhibits dimer formation and DNA binding properties identical with those of the wild-type repressor. Moreover, the 5-OHTrp provides a spectral signal that allows monitoring of the protein concentration without interference from DNA. In this article, the ability to selectively detect 5-OHTrp-labeled repressor during analytical ultracentrifugation is used to study the higher order assembly of repressor dimers in the absence and in the presence of operator DNA. Contrary to the expectation that tetramer might be the limiting oligomer, lambda cI repressor undergoes a definite association to octamer. The relatively narrow concentration range over which transition from predominantly dimer to predominantly octamer occurs makes it unlikely that significant levels of tetramer are formed in the absence of DNA. Moreover, mass measurements reveal that an OR1 oligonucleotide binds to octameric repressor and does not dissociate it to tetramers. The use of the 5-OHTrp spectral enhancement opens a promising new avenue for the exploration of protein-protein and protein-nucleic acid interactions by analytical ultracentrifugation.
Laue
T M
TM
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham 03824.
Senear
D F
DF
Eaton
S
S
Ross
J B
JB
D006916:5-Hydroxytryptophan; D010582:Bacteriophage lambda / Q000378:metabolism*; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; D006916:5-Hydroxytryptophan