Fluorescence analysis of the Escherichia coli transcription regulator H-NS reveals two distinguishable complexes dependent on binding to specific or nonspecific DNA sites.
Here we report a structural investigation of the transcription factor H-NS and its DNA interaction. H-NS has a general effect on transcription by compacting DNA; but for a number of specific genes, it is known to act directly as repressor or activator. The homodimeric protein binds to the major groove of DNA in a sequence-nonspecific manner, recognizing a curved conformation of the target DNA. H-NS consists of 136 amino acids with a single tryptophanyl residue at position 108. To overcome the apparent lack of any other structural details, we took advantage of the intrinsic fluorescence of Trp-108. Static and dynamic quenching constants obtained with the neutral quencher molecule acrylamide are consistent with a hydrophilic environment and high degree of solvent exposure for Trp-108. In addition, quenching studies in the presence of the anionic quencher iodide indicate a positively charged microenvironment for the same amino acid residue. Specific and nonspecific H-NS.DNA complexes were studied by gel retardation and fluorescence analysis. While specific H-NS.DNA complex formation is accompanied by a clear enhancement of the tryptophanyl fluorescence intensity, interaction in the presence of the nonspecific competitor DNA poly(dI-dC) decreases the fluorescence quantum yield.
Tippner
D
D
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Federal Republic of Germany.
Wagner
R
R
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D004926:Escherichia coli; D007454:Iodides; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; C031908:H-NS protein, bacteria; D007454:Iodides; D012335:RNA, Ribosomal; D014157:Transcription Factors