Transcription by RNA polymerase serves as the key
regulatory step in bacterial gene expression. The core RNA
polymerase is a protein complex consisting of 5 subunits
(two alpha, beta, beta', and omega) and is capable of
synthesizing mRNA off of a DNA template. However a sixth,
modular subunit known as the sigma factor is required to
melt the dsDNA and provide a single stranded DNA template
for transcription. When sigma factor binds to the core RNA
polymerase the complex is known as the RNA polymerase
Most bacteria contain multiple sigma factors each
responsible for regulating the transcription of a subset
of genes. Sigma factors are further subdivided into the
sigma70 and sigma54 classes,
though there is no sequence homology between the two.
Members of the sigma70 class of sigma factors are
immediately active upon binding to the DNA and the core
RNA polymerase, so transcription initiation occurs as soon
as sigma70 proteins are translated. Regulation of sigma70
class sigma factors is primarily achieved through changing
the sigma70 expression levels and the expression of
anti-sigma factors that bind to and inactivate them.
Members of the sigma54 class require an additional
ATP-dependent activation event provided by one of a number
of AAA+ ATPase transcriptional
activators before they can melt dsDNA and initiate
transcription. This activation requirement gives
sigma54 transcription a tighter control over gene
expression and a wider dynamic range. As a consequence,
sigma54-dependent gene expression is often responsible for
creating swift and precise responses to environmental
Mechanism of Transcription Initiation by σ54
Transcription initiation with sigma54 differs from
transcription initiation by other sigma factors due to its
additional activation requirement. Transcription
initiation is achieved after:
the components (RNAP, sigma54, activators) on the DNA
upstream of the gene
the sigma54 subunit by the AAA+ ATPase activators
the DNA by the RNAP holoenzyme (core RNAP along with
the sigma54 subunit)
transcription of RNA off of the template DNA strand
These four stages are depicted in the figure below.
cartoon depiction of sigma54 (green) binding to DNA
(yellow), core RNA polymerase (subunits in shades of
blue) and a transcriptional activator/enhancer (subunits
in shades of red).
Assembly: The AAA+ ATPase
transcriptional activators (such as NtrC in E. coli)
bind as dimers at enhancer sequences ~100bp upstream of
the gene start site. Two sets of dimers assembled at the
enhancer sequence along with a third dimer in solution
will assemble into the active conformation, a hexameric
ring capable of hydrolyzing ATP. Integration Host Factor
(IHF) binds in between the enhancer sequence and sharply
bends the DNA bringing the transcriptional activator
into close contact with sigma54.
The sigma54-RNAP holoenzyme assembles on a conserved
promoter sequence at the -24 site through specific
interactions with its C-terminal RpoN box motif. This is
shown below for Aquifex aeolicus sigma54 and the gene
nirB (and discussed in more detail in the -24 DBD
section further down).
Activation: When the components have
assembled, the hexameric transcriptional activators
contact the N-terminus of sigma54. A conserved GAFTGA
motif near the central pore of the hexamer is essential
for this contact. At least one ATP hydrolysis cycle is
also a requirement for activation. Little is understood
about the structural changes to sigma54 brought about by
the transcriptional activators, but while sigma54 can
assembly on the DNA before activation it is only capable
of opening the DNA after activation.
DNA Opening: Activated RNAP holoenzyme (core
RNAP plus the sigma54 subunit) is capable of opening DNA
near the -12 promoter sequence. The sigma54 -12 DNA
binding domain has been shown to interact with single
stranded DNA (trapped in a bubble like open complex) and
likely plays a large role in opening the DNA.
Elongation: Once open, core RNAP begins to
elongate RNA off of the DNA template starting from the
+1 gene site. Core RNAP alone is able to transcribe RNA
off of opened DNA, but is unable to open DNA without the
help of activated sigma54. At some point after DNA
opening, sigma54 falls off the core RNAP enzyme and
In the Wemmer Lab, we study the components of sigma54
transcription initiation using a combination of NMR
spectroscopy, X-ray crystallography, Short Angle X-ray
Scattering, and other biochemical and biophysical
techniques. By probing the structure and dynamics of these
proteins we hope to better understand how sigma54
interacts with and regulates communication between the
DNA, core RNA polymerase, and the AAA+ ATPase activators,
and the overall mechanism of bacterial transcription
initiation by sigma54.
Functional domains of σ54
The sigma54 class of sigma factors has traditionally been
divided into three regions with the N-terminal ~60
residues termed Region I, followed by a linker domain
termed Region II, and a large C-terminal region with
multiple individually folding functional domains (from ~69
to 398 in A. aeolicus) termed Region III. With the
results of recent structural studies, a more intuitive way
to divide sigma54 is into four functional domains. A
schematic of these domains are shown for the thermophile
Aquifex aeolicus in the figure below:
Activator Binding Domain
(ABD): The N-terminal domain (residues 1-56) of
sigma54 (often referred to as Region I) interacts with the
conserved GAFTGA motif in the central AAA+ ATPase domain
of a variety of transcriptional
activators. This interaction serves as a switch for
sigma54 activation and leads to a conformational change
that allows sigma54 to switch from a closed to an open
complex with DNA, the step at which double stranded DNA
melts allowing core RNA polymerase to transcribe mRNA off
of the single stranded DNA template.
After the Activator Binding Domain comes a variable length
linker region. In A. aeolicus the linker region is
relatively short compared to most other homologs.
Core Binding Domain (CBD):
Following the linker domain is a seven-helix domain that
interacts with the core RNA polymerase. It can be further
split into two sub-domains: a four helix bundle (residues
69-135) and a three-helix bundle (residues 135-198). A
structure of the Core Binding Domain of A. aeolicus was
solved by NMR in the Wemmer Lab. It represents a
conformational "fracture point" that could be responsible
for transferring the force of activator binding and ATP
hydrolysis into larger conformational changes in the
C-terminal sigma54 DNA binding domains to promote the
formation of an open complex with DNA and transcription
Ensembles of the 20
best solution structures (stereo) determined
from NMR data for sigma54 (69–198). PDB: 2K9M,
-12 DNA Binding Domain
(-12DBD): The next domain is known to interact
with the -12 DNA element 12 base pairs upstream of the
transcription start site.[6,7] Once sigma54 is activated,
this domain is heavily involved in melting the DNA to
initiate transcription. However, structural and dynamic
information about this protein-DNA interaction is limited.
-24 DNA Binding Domain (-24DBD): The C-terminal
domain binds DNA at a conserved -24 DNA element upstream
of the transcription start site. Unlike the -12DBD
which seems to play an active role in melting the DNA
strand, the -24DBD serves to position and anchor the
inactive sigma54 factor at the proper location along the
DNA. A structure of the -24 DNA Binding Domain of A.
aeolicus was solved by NMR in the Wemmer Lab.
NMR Structure of the
sigma-54 DNA-Binding domain from Aquifex
aeolicus at different stages of structural
refinement. Blue=20 NOEs assigned, marine=200,
cyan=400, lime=444, green=535, final RMSD 0.635
Å. (PDB: 2O8K and 2O9L)
Current work in the Wemmer Lab focuses on filling in the
details of various domains of A. aeolicus sigma54 not yet
characterized and expanding upon our current structural
knowledge. A more complete picture of sigma54 will help us
understand how sigma54 interacts with DNA, core RNA
polymerase, and the AAA+ ATPase transcriptional activators
as well as the broader mechanism of sigma54 transcription
Mechanism of σ54
activation by AAA+ ATPases
We employ a variety of biophysical and biochemical
techniques to study the mechanism sigma54 activation by
NtrC-like transcriptional activators. In addition to
probing interactions between the two, current work in our
lab also focuses on studying various states of the
transcriptional activators themselves. More information
about our lab's work on AAA+ ATPase transcriptional
activators can be found here.
 Wedel, A., Kustu, S. (1995) The bacterial
enhancer-binding protein NTRC is a molecular machine: ATP
hydrolysis is coupled to transcriptional activation. Genes
Dev. 9, 2042-52.
 Kustu, S., E. Santero, J. Keener, D. Popham, and D.
Weiss. 1989. Expression of sigma 54 (ntrA)-dependent genes
is probably united by a common mechanism. Microbiol. Rev.
 Kazmierczak, M.K., Wiedmann, M., Boor, K.J. (2005)
Alternative Sigma Factors and Their Roles in Bacterial
Virulence. Microbiol. Mol. Biol. Rev. 69, 527-543.
 Buck, M. Gallegos, M.-T., Studholme, D.J., Guo, Y.,
Gralla, J.D. (2000) The Bacterial Enhancer-Dependent sigma
54 (sigma N) Transcription Factor. J Bacteriology. 182,
 Hong, E., Doucleff, M. & Wemmer, D.E. Structure of
the RNA polymerase core-binding domain of sigma(54)
reveals a likely conformational fracture point. Journal of
molecular biology 390, 70-82 (2009).
 Barrios, H., Valderrama, B., Morett, E. (1999)
Compilation and analysis of sigma(54)-dependent promoter
sequences. Nuc. Ac. res. 27, 4305-13.
 Wong, C., Tintut, Y., Gralla, J. (1994) The Domain
Structure of Sigm54 as Determined by Analysis of a Set of
Deletion Mutants. J. Mol. Biol. 236, 81- 90.
 Doucleff, M. et al. Structural basis of DNA
recognition by the alternative sigma-factor, sigma54.
Journal of molecular biology 369, 1070-8 (2007).
 Wigneshweraraj, S. R., P. C. Burrows, et al. (2005).
The second paradigm for activation of transcription.
Progress in Nucleic Acid Research and Molecular Biology,
Academic Press. Volume 79: 339-369.