Allosteric control of cAMP receptor binding dynamics.

Area of Science:

• Molecular Biology
• Biophysics

Background:

• The cyclic AMP receptor protein's (CRP) interaction with DNA is crucial for gene regulation.
• Intrinsic fluorescence of CRP is a sensitive probe for DNA binding, but susceptible to photoreaction artifacts.

Purpose of the Study:

• To accurately quantify CRP-DNA reaction kinetics using fluorescence.
• To elucidate the role of cAMP concentration in CRP binding to promoter DNA.

Main Methods:

• Utilized a ratio procedure to correct for photoreaction-induced signal perturbations.
• Measured CRP-DNA binding kinetics across a range of salt and pH conditions.
• Investigated the effect of cAMP concentration on CRP binding to promoter DNA.

Main Results:

• Corrected fluorescence transients revealed broad time-range CRP-DNA reactions at various salt concentrations.
• Initial CRP binding is rapid and similar for specific and nonspecific DNA at low salt (13.5 mM, pH 7).
• CRP-DNA binding kinetics are salt and pH-dependent, with slow steps observed up to hundreds of seconds.
• CRP-promoter DNA binding requires two cAMP molecules per dimer, with a rate constant of 1.3 × 10(8) M(-1) s(-1).

Conclusions:

• A ratio correction method effectively quantifies CRP-DNA interactions.
• CRP binding to promoter DNA is a multi-step process influenced by salt, pH, and cAMP levels.
• The binding stoichiometry necessitates two cAMP molecules per CRP dimer for high-affinity promoter binding.