HENRY M. SOBELL

A MECHANISM TO UNDERSTAND THE FORMATION OF THE RNA
POLYMERASE-PROMOTER TIGHT BINDING COMPLEX

The presence of premeltons undergoing low amplitude breather motions within promoter regions can serve the important function of providing nucleation centers for site-specific DNA melting by the RNA polymerase enzyme (and, possibly, by other proteins that play a role in gene activation).

One can envision the formation of the transcriptionally competent tight binding complex to involve the initial attachment of the polymerase to a premelton located at or near the start of transcription (shown on the left), triggering a cascade of conformational changes in both the polymerase and the DNA (shown in the middle), that lead to the formation of the tight binding complex (shown on the right).

The process described above can be considered to be a series of concerted allosteric transitions that result in the progressive union of two molecular species. How might this occur, and what is its underlying energetics?

This process is best understood as a protein-DNA structural phase transition, the emergent phase being the RNA polymerase-promoter tight binding complex. Complex formation entails a series of stepwise conformational transitions, in which energy is transferred from the polymerase to the DNA in the form of small packets (i.e., "kinks"). This is possible, providing the protein begins in a high-energy metastable state. It can then spontaneously fall into lower lying metastable states, as DNA melting and tight complex formation ensue. Such an adiabatic process is expected to have little (or no) net change in free energy.

The mechanism is reversible. One can imagine the transcriptionally competent tight binding complex (shown on the right) to undergo a series of concerted allosteric transitions (shown in the middle) that lead to the final detachment of the polymerase from the premelton (shown on the left). Such a mechanism necessarily accompanies the termination of transcription at the 3' ends of genes.

The level of negative superhelical strain energy in DNA provides the bias that determines the direction of this protein-DNA phase transition. Other more active processes can also be involved.

It is well known that the transcriptionally competent RNA polymerase-DNA complex is associated with an extremely large (apparent) binding constant. Classical thermodynamics would predict a large net negative free energy change to accompany the binding reaction. How then is it possible for the RNA polymerase to move along DNA during the process of DNA transcription?

This is understood in the following way. The binding by the RNA polymerase to the promoter is an adiabatic process, energy being transferred from the protein to the DNA in a series of stepwise allosteric transitions that lead to the formation of the transcriptionally competent tight-binding complex (see above). Although there is little or no net free energy change expected for such a process (this being an example of a protein-DNA structural phase transition), the final structure contains both molecular species topologically linked together (i.e., in a way analogous to how a white AVEC easy zipper is attached to the tracks on a "Ziploc" plastic bag). Such a model predicts the transcriptional complex to be able to "slide" with minimal friction along DNA during transcription, in spite of the large apparent binding constant holding these molecular species together. This model accounts for the processivity observed in RNA synthesis as well.

The tight-binding transcriptionally competent complex arises as the result of topological linking (i.e., "intertwining"), not from the presence of a large negative free energy change accompanying complex formation. The apparent binding-constant for this complex, therefore, should not be confused with a true equilibrium-binding constant, as described by classical thermodynamics.