RESEARCH



Research in our laboratory is focused on interactions between DNA, RNA and proteins. Our goal is to understand the step-wise structural and energetic changes associated with these binding events. Dye molecules attached to oligonucleotides and proteins act as reporter groups, sensitively signaling structural changes that occur on the nanosecond time scale. We thus make extensive use of laser-induced fluorescence to monitor protein-oligonucleotide interactions in real time.

A particularly powerful fluorescence technique is fluorescence resonance energy transfer (FRET), in which there is a non-radiative transfer of energy between a donor dye molecule and an appropriate acceptor molecule. The efficiency of energy transfer is highly dependent upon the distance between the donor and acceptor, allowing determination of distances and distance changes from 40 to 80 Å with precision well within 1 Å. The 14-mer oligonucleotide shown below has a fluorescein on the 3' end that functions as a FRET donor and a rhodamine derivative on the 5' end that functions as an acceptor in energy transfer.

 

We use such molecules to:

  • monitor conformational changes induced in DNA upon protein binding
  • determine the complex recognition mechanisms of various protein-DNA interactions, using real-time kinetic measurements
  • detect single nucleotide mismatches in oligonucleotides and in plasmid DNA
  • measure the kinetics of DNA hybridization.




Function Studies

In eukaryotes, transcription (the initial step in protein synthesis) begins with the binding of TBP (TATA binding protein) to a unique promoter sequence termed the "TATA box". The DNA sequence shown above contains the TATA sequence from the consensus Adenovirus major late promoter, TATAAAAG. Upon binding of TBP to DNA bearing the AdMLP promoter, one pair of phenylalanines intercalates between bases 1 and 2 and another pair between bases 7 and 8, inducing sharp kinks at these sites and unwinding the DNA between bases 2 and 7. The result is an 80° bend in the DNA.

• We have monitored TBP-AdMLP association and dissociation binding in real time using fluorescence stopped-flow and have shown that this binding process is complex. The simplest model consistent with the data is:


A small population of pre-bent DNA binds to the TBP, with two intermediate conformers apparent along the pathway to formation of the final TBP-DNA complex. The first intermediate species remains at significant mole fraction at equilibrium. Note that all three bound forms are in equilibrium. This model is consistent with molecular-dynamics simulations showing significant populations of bent free DNA duplexes. We are investigating the release of water molecules associated with the multi-step binding.

The multi-step, simultaneous binding and bending is depicted in the following cartoon animation:

 


• TBP recognition of a second consensus promoter, E4 (TATATATA), also corresponds to a linear 3-step model. TBP binds tightly to E4 and bend it ~80f, as with AdMLP. Despite similar overall thermodynamics, the step-wise enthalpic and entropic compensations differ significantly for TBP binding these consensus TATA sequences.

• In contrast to the consensus sequences, TBP recognition of a naturally occurring variant promoter (TATAAACG) is described by a 3-step model with two branching pathways. One pathway proceeds through an intermediate having severely bend DNA, reminiscent of the consensus reactions. The other branch yields a unique conformer with shallowly bent DNA. The resulting TBP•TATA complex has moderately bent DNA and diminished relative transcription activity.

Solution Structure Studies

• The distance between donor and acceptor moieties in double-labeled TBP-bound DNA has been determined to very high precision using FRET. The DNA bend angle may then be determined in terms of simple models. The bend angle thus determined for bound AdMLP in solution, 77°, is in excellent agreement with the 80° bend determined in high resolution crystallographic studies.

However, single point mutations in the AdMLP TATA box produce profound changes in the DNA bend angle and in the kinetics of DNA-TBP association. The solution bend angles determined in our laboratory using FRET are 80° for a consensus sequence but range from 32° to 63° for five variant sequences. These solution bend angles correlate very well with the efficiency of in vivo and in vitro transcription. We hypothesize that the DNA bend angle in the binary DNA-TBP complex determines the positioning of subsequent proteins that must bind appropriately for efficient transcription.

• In contrast to the sequence-dependent bend angles determined in our laboratory, co-crystals of consensus and 12 variant sequences bound to various TBP molecules all have 80° bends. Crystals of DNA-TBP complexes are grown from solutions of osmolytes, such as glycerol and ethylene glycol, which lower the activity of the water, facilitating crystallization but also facilitating loss of water at the TATA-DNA interface. We have shown that these osmolytes induce profound increases in the solution DNA bends of two transcriptionally active TBP-bound variant sequences to a common angle of 80°. These osmolyte effects reconcile the solution and crystal structural differences and are summarized (in degrees) in the table below:

• In work unrelated to DNA-TBP interactions, DNA bending by various anti-tumor agents is being investigated using fluorescence methods for comparison with bending determined from the efficiency of cross-linking.


Other ongoing work includes investigations ofS

  • DNA bending by various anti-tumor agents using fluorescence methods, for comparison with bending determined from the efficiency of cross-linking.

  • the conformation and flexibility of DNA duplexes in solution.




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RESEARCH