The
changes in
heat capacity (deltaCp)
that occur upon folding of biomolecules are often quite significant. In
protein folding this phenomenon is quite well studied, but it has for
the most part been ignored in the study of nucleic acids. This
parameter is most often equated with changes in solvent structure or
hydration around the macromolecule between the native and unfolded
states. In extreme cases where the deltaCp term is large, the
phenomenon of cold denaturation can be observed because the deltaCp
term contributes significantly to the overall folding free energy at
low temperature.
Heat capacity changes can be defined based on either the temperature
dependence of the enthalpy or entropy of folding. Experimentally, we
typically use the temperature dependence of the folding enthalpy in
that it is readily measured by isothermal titration calorimetry.
 |
 |
Figure 1. Plot of deltaG versus
temperature based on the Gibbs-Helmoholtz equation showing the effect
of a large deltaCp on the overall deltaG.
|
Figure 2. Isothermal titration
calorimetry allows us to follow nucleic acid folding of bimolecular
constructs. We use this methodology to obtain the heat capacity changes
by repeating the titration at several temperatures.
|
Non-coding
RNAs
(ncRNA) are small conserved RNA molecules that participate in a variety
of regulatory pathways in cells. We have been studying the small ncRNAs
involved in post-transcriptional gene regulation in bacteria -
primarily in the control of stress response pathways. By using a
riboregulatory circuit wherein genes are controlled at the
post-transcriptional level by small RNAs, bacteria have developed very
rapid mechanisms to turn on and off pre-programmed stress responses
that require much less transcription during this critical period.
About half of these
ncRNAs bind to a small, hexameric protein
called Hfq which is a bacterial homolog of the eukaryotic Sm proteins.
This protein is known to facilitate the interactions between ncRNAs and
their cognate mRNAs (see figure). While
hfq is not an essential gene,
bacteria engineered to be
hfq-
display a variety of stress sensitive phenotypes.
The regulation of a given mRNA can result from three possible outcomes
upon expression of the ncRNA. The mRNA can become translationally
activated, it can become translationally repressed or it can be
targeted
for degradation. The generic model for how translational activation
works is shown schematically below where an element of structure
occludes either the ribosome binding site or the start codon or both of
them. Upon refolding due to the interaction with the ncRNA, these
sequences become accessible and translation begins.
Using DsrA and rpoS mRNA as a model (secondary structures shown below),
we have been studying how Hfq functions. We wish to understand how Hfq
facilitates the proper RNA-RNA partnerships without becoming
kinetically trapped in a complex mixture of non-functional states. We
have addressed this problem by studying the binary and ternary
complexes Hfq makes with ncRNA and mRNAs. Our approach melds in
vitro
biochemical and biophysical techniques with bacterial genetics and in
vivo analysis using reporter constructs. Using these tools, we recently
showed that Hfq binds different RNAs on each face of the torus. These
data support a model wherein the non-coding RNA lies on one face of the
protein with the mRNA wrapping around to make contacts with both faces.
These interactions thus present the two RNAs to one another which
facilitates proper base pairing when the appropriate complementary
sequences are present.
 |
 |
Figure 3. A network of regulatory
interactions governed by Hfq. Hfq has been shown to bind each of the
ncRNA (circled) and mRNAs (outer ring) facilitating the
post-transcriptional gene regulation that responds to the indicated
environmental stress.
|
Figure 4. Generic model through
which regulatory ncRNAs can relieve translational repression of a
complementary mRNA through strand displacement. Equivalent models can
be used to explain translational repression as well.
|
Clostridium
difficile is the organism responsible for antibiotic associated
diarrhea, a common problem resulting from the activity of two exotoxins
produced by this ubiquitous enteric bacterium. Toxins A and B are
glucosyltransferases that catalyze the modification of the RhoA
sub-family of G-proteins in a Mn(II)/Mg(II) dependent reaction. We are
analyzing the mechanism of these metalloenzymes through kinetic and
biophysical studies with the ultimate goal of developing highly
specific inhibitors for these toxins.
 |
Figure 7. C. difficile bacterium forming an
endospore.
|
