σ54 Regulated Two-Component
Signal Transduction Systems
Two-component signal transduction systems are a common
prokaryotic means for sensing environmental stimulus and
providing appropriate cellular response. In NtrC systems
the first component (a histidine kinase) senses an
environmental change and auto-phosphorylates. The
phosphoryl signal is transferred to the three-domain
transcriptional activator (also referred to as response
regulators and enhancer binding proteins). About half of
all transcriptional activators, including most of those
studied in our lab, are regulated by an N-terminal
receiver domain, which accepts the phosphate signal. They
also have a central ATPase domain, which oligomerizes upon
activation and, through an ATP hydrolysis coupled
mechanism, induces a conformational change in sigma54 that
renders it competent for initiating transcription.
Finally, they have a C-terminal DNA-binding domain which,
among other functions, positions the activator upstream of
the desired gene. We study the structure and mechanisms of
these transcriptional activators using a combination of
NMR spectroscopy, X-ray crystallography, Short Angle X-ray
Scattering, and other biophysical techniques, in order to
better understand the process of bacterial transcription
and the mechanism of these AAA+ ATPases.
Two-component signal transduction pathway in σ54
Natasha Keith Vidangos's thesis, 2009
A histidine kinase senses an environmental
change and auto-phosphorylates on a conserved
histidine. The phosphate is transferred to a
conserved aspartate on the receiver domain of one
of a number of transcriptional activators that are
bound as dimers about 100bp upstream of the
transcription initiation site. Other regulatory
domains besides the receiver domain have similar
Once the receiver domain receives the signal, the
transcriptional activator, whether by a positive
or negative regulatory mechanism, no longer favors
the dimeric state. The central domain of these
transcriptional activators oligomerize into a
hexameric or heptameric ring, which functions as a
AAA+ ATPase. The activated oligomer, still
bound 100bp upstream of the initiation site, is
brought into contact with sigma54 by DNA bending.
transcriptional activator contacts the N-terminal
domain of sigma54
its highly conserved GAFTGA loop. Through ATP
hydrolysis and an unknown mechanism, the
transcriptional activator interacts with sigma54.
Sigma54 then undergoes a conformational change
that enables it to melt the dsDNA at the site of
transcription (initiation). Once the DNA is
melted, core RNA polymerase begins transcribing
mRNA off of the DNA (elongation).
Structural studies of transcriptional activators of σ54
The current structural information of these AAA+ ATPase
transcriptional activators (also known as enhancer binding
proteins) is represented in the figure below:
Circular regulatory domains indicate two-component
receiver domains. Star-shaped regulatory domains
indicate GAF domains. Color coding is as follows: red
indicates a high-resolution structure is available;
green indicates multiple high resolution structures are
available; blue indicates a low-resolution structure is
Recent structures to come out of the Wemmer Lab include
the NtrC1 active and inactive state, as well as the NtrC4
inactive state and the NtrC4 DNA binding domain. These
structures, along with other biochemical and biophysical
studies, have contributed to our understanding of various
NtrC-family regulatory mechanisms.
|In NtrC1 (right),
the presence of its receiver domain represses
assembly of the oligomeric protein complex until
activated by ATP (or the ATP mimic BeF3) which
disrupts the dimeric interface between two
regulatory domains thus allowing oligomerization.
When removed altogether, the NtrC1-C central
domain alone but at high enough concentrations
will spontaneously oligomerize into a AAA+ ATPase
domain capable of hydrolyzing ATP. Because the
receiver domain serves to inhibit formation of the
active oligomer, NtrC1 is said to be negatively
regulated. This is in contrast to the
positively regulated homologue NtrC (left),
in which the active AAA+ ATPase oligomer is
promoted by the presence of the N-terminal
receiver domain and will not assemble at all
without it. In NtrC4 (middle),
the unactived receiver domain also represses
assembly of the hexamer like in NtrC1, but to a
much lesser extent. This suggests that NtrC4
represents an evolutionary intermediate between
the positive and negative regulatory mechanisms of
NtrC and NtrC1 respectively.
Below are the three recent structures to come out of the
Wemmer Lab that have helped explain the regulatory
mechanisms of these NtrC-like proteins.
|(a) The NtrC4-RC inactive
state (PDB: 3DZD), (b)
the NtrC1-RC inactive state (PDB: 1NY5)
and (c) the NtrC1-C active state (PDB: 1NY6) all
solved by X-ray Crystallography in the Wemmer
Lab. The inactive state receiver domains both
form dimers. The NtrC1 central domain alone
enters its active state and forms a heptameric
ring when its receiver domain is removed.
DNA binding in NtrC-family proteins
The C-terminal DNA binding domain in all NtrC-family
proteins interacts with a promoter DNA sequence upstream
of the transcription start site. One function of the DNA
binding domain is to colocalize the transcriptional
activators near the sigma54 promoter site, raising the
local concentration of the activator and thus increasing
the probability of sigma54 activation. A second
function is to localize NtrC-family proteins nearby one
another to speed up the oligomerization process of the
NtrC proteins upon activation.[15,16] The two DNA binding
sites exhibit cooperative binding, contributing further to
this effect. A third function of the DNA binding
domain may be to stabilize the off-state dimer, although
this isn't necessary for NtrC1 and NtrC4 dimerization,
perhaps because of the strong dimerization interface
formed by their negatively regulated receiver domains.
Recent work in the Wemmer Lab has characterized the
structure of the DNA binding domain of NtrC4 in complex
structure of the NtrC4 DNA Binding domain in
complex with DNA. The NtrC4 DBD dimerizes and
folds into a helix-turn-helix motif that binds
about 100 base pairs upstream from the
transcription start site. The complex reveals
specific interactions in the major groove of DNA
as well as non-specific interactions with the DNA
backbone. The structure shows NtrC4 DBD creates a
very slight bending of the DNA, in contrast with
related proteins like Fis where the DNA is bent
40-90 degrees. This may reflect an evolutionary
trend that has eliminated the need for DNA bending
or something specific to the thermophilic nature
of NtrC-like proteins in A. aeolicus.
The Wemmer Lab is currently working to fill the gaps in
our structural understanding of NtrC family proteins in
various states in order to better understand how these
proteins interact with each other, the upstream DNA and
the sigma54 factor itself.
Other questions about NtrC family transcriptional
NtrC-like proteins, NtrC1 and NtrC4, belong to a special
subfamily of response regulators that activate
transcription via interaction with sigma54 of the RNA
polymerase holocomplex. In addition to the structural
characterizations, in the Wemmer Lab we are also
investigating a number of NtrC-related questions
- How is the activation signal transmitted
intramolecularly? How are the DNA promoter sites
- How is this recognition different in thermophilic
- How do NtrC-like proteins signal sigma54 and
activate it for transcription initiation?
- How do NtrC homologues and paralogues differ
functionally and structurally?
Structural studies of σ54
In the Wemmer Lab, we also study the structure and
function of the sigma54 factor, including the structure of
its individual domains, the structural requirements for
function, and the DNA-binding activity. For more
information see our σ54
structural studies page.
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