Specificity, stability and mechanisms of formation of
protein-nucleic acid complexes; biophysical studies of the E. coli
cytoplasm; polyelectrolyte properties of nucleic acids and their
complexes
Site
specific interactions between DNA binding proteins and their target
sequences govern the expression and replication of genetic information.
To understand these central noncovalent binding processes, our effort
is focused on quantifying the thermodynamics (energetics) and kinetics
of interaction between DNA and three bacterial proteins: RNA
polymerase, lac repressor, and ?integration host factor? (IHF). All
these systems are unified by a common theme: large conformational
changes and other coupled processes in the proteins and/or their target
DNA sites occur in binding. To initiate transcription from promoter DNA
sites, RNA polymerase opens more than 10 base pairs of the DNA helix in
the vicinity of the transcription start site, and in the process
creates the catalytic site for NTP binding and synthesis of the RNA
transcript. Lac repressor folds alpha helices in the minor groove of
its target DNA sequence and wraps or loops flanking DNA regions to act
as an on-off switch for transcription of genes for growth on the sugar
lactose. To wrap and package DNA, IHF induces a large bend (>160?)
in its specific binding site.
We use a wide range of
biophysical and biochemical measurements to characterize these
conformational changes and to quantify the amount of biopolymer surface
they expose to or remove from solvent and solutes. From thermodynamic
and kinetic studies, we determine the balance between driving forces
and free energy costs for these conformational changes, and
characterize the sequence of mechanistic steps by which they occur. We
also study the DNA binding behavior of oligocations and model proteins
to dissect contributions from individual components of the overall
protein-DNA binding surface, and do computational and analytic theory
to describe the behavior of these simpler systems.
A summary of our work defining the series of conformational changes
orchestrated by RNA polymerase in the mechanism to form the open
promoter complex and the transcription bubble is shown below in
proposals which incorporate literature structural data for free
polymerase. The three step mechanism (Fig. 1), based on our kinetic
studies and low resolution structural data for the intermediates (from
chemical and enzymatic footprinting) postulates large scale changes in
each step including DNA wrapping, kinking, unpairing and unstacking as
well as protein folding and hinge bending (jaw closing). We propose
that the first kinetically-significant intermediate I1;
( Fig. 2) has a sharp bend upstream of the transcription start site
which puts the downstream DNA in the jaws of polymerase prior to
opening.
Current
work in our lab is characterizing these conformational changes and
coupled processes (including coupling of disruption of protein surface
salt bridges to DNA wrapping) by thermodynamic, kinetic and
footprinting methods, using selected protein structural variants and
DNA sequence variants and analyses based on the recent crystal
structures of eucaryotic, prokaryotic and phage RNA polymerases.
Other projects in the laboratory include the characterization of the
bacterium E. coli as a chemical and osmotic system, and the
thermodynamic and molecular characterization of interactions of
cytoplasmic solutes (e.g. potassium glutamate, glycine betaine) and
common biochemical solutes (e.g. urea, glycerol) with biopolymers and
of effects of these solutes on biopolymer processes.
Graduate students from Biochemistry, Chemistry and Biophysics are
conducting this research. The broad range of backgrounds and interests
of these students has been a key factor in our research successes and
contributes to a stimulating research environment. Many of my students
have gone on to academic positions in chemistry and biochemistry
departments; many others are engaged in research at chemical,
pharmaceutical and biotechnology companies