Fluorescence studies of a single tyrosine in a type II DNA binding protein

Abstract

We studied the fluorescence properties of a single tyrosine (Tyr94) located in the C-terminal tail of transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by the Bacillus subtilis phage SPO1. The time-resolved fluorescence intensity of Tyr94 in free TF1 dimers decays as a single exponential, and this is consistent with a twofold symmetrical structure. The fluorescence is readily quenched by acrylamide, but it is less accessible to anionic quenchers (iodide and citrate), suggesting that the tyrosine is located on the protein surface in a negatively charged environment provided by neighboring Glu95 and Asp96 residues. TF1 dimers associate at moderate concentrations (greater than 0.02 mg/mL) as judged from concentration dependencies in the molar fluorescence intensity, the steady-state fluorescence polarization, and the bimolecular quenching constants. Nonspecific binding of TF1 to SPO1 and calf thymus (CT) DNA and various double-stranded polynucleotides quenches the Tyr94 fluorescence to varying extent. Fluorescence lifetimes of TF1 in the bound state correlate with spectral overlaps between TF1 emission and DNA absorption, demonstrating that excitation energy transfer to DNA bases contributes significantly to the observed quenching. From analysis of the observed quenching in the DNA complexes we conclude that Tyr94 is located within 10-14 A of the DNA helix axis and not in direct contact with the DNA bases. Equilibrium analyses based on fluorescence titrations show that the maximum binding density on DNA extrapolates to ca. 1 TF1 dimer/5 DNA base pairs. We find several differences in TF1 binding to SPO1 DNA, which contains hydroxymethyluracil instead of thymine, and CT DNA: (i) The tyrosine residue is less exposed to the solvent in the SPO1 DNA complex than in the CT DNA complex. (ii) D2O addition enhances the Tyr94 fluorescence when TF1 binds to SPO1 DNA but not when it binds to CT DNA. (iii) The TF1-SPO1 DNA complex is stable at higher NaC1 concentrations than is the TF1-CT DNA complex, and its formation involves the dissociation of more Na+ ions than does the TF1-CT DNA complex. On the basis of these observations and the fact that the Tyr94-containing tail of TF1 is essential for binding to SPO1 DNA, we discuss various models for the TF1-DNA complex.

Determination of DNA‐binding parameters for the Bacillus subtilis histone‐like HBsu protein through introduction of fluorophores by site‐directed mutagenesis of a synthetic gene

“…In contrast to the findings of Watanabe et al (1984), a dimer of HBgl protein has been suggested to contact 3.5 bp of DNA (Imber et al, 1982). In addition, the B. subtilis phage SPOl-encoded TF1 protein appears to contact 5 bp of its 5-hydroxymethyluridine-containing DNA (Hard et al, 1989). I t has recently been shown by calorimetry and circular dichroic spectroscopy that HBsu protein can undergo structural changes depending on ionic strength and protein concentration (Welfle et al, 1992).…”

Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1

“…1). It is known from previous work that binding to SPOI DNA brings Tyr-94 of TF1 into the proximity of this DNA (11). A recent nuclear magnetic resonance (NMR) analysis has also revealed that, in the free TF1 dimer, Phe-97 in the tail is positioned close to Phe-61 in the putative DNA-binding arm (24).…”

Proton and nitrogen NMR sequence-specific assignments and secondary structure determination of the Bacillus subtilis SPO1-encoded transcription factor 1

“…The Phe* variant contained as its sole added protiated residue, [2,4-2H2] phenylalanine, synthesized according to a procedure to be discussed elsewhere (Dauzonne & Parello, in preparation). Procedures for the purification ofTFl fromMG1655(pTFlX), have been described previously (Reisman et al, 1993;Hard et al, 1989a). When similar procedures were used to obtain protein from DL39(pTFlX), we used a medium described by Muchmore et al (1989), containing appropriate unlabeled amino acids for growth and labeled amino acids for over expression.…”