Transcription Initiation by σ54

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 holoenzyme.

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.[1] 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 change.[3]

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:
  1. Assembly of the components (RNAP, sigma54, activators) on the DNA upstream of the gene
  2. Activation of the sigma54 subunit by the AAA+ ATPase activators
  3. Opening of the DNA by the RNAP holoenzyme (core RNAP along with the sigma54 subunit)
  4. Elongation and transcription of RNA off of the template DNA strand
These four stages are depicted in the figure below.

A 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).

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.

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 elongation continues.

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.[4] 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.

Linker region: 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.[5] 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 initiation.

Ensembles of the 20 best solution structures (stereo) determined from NMR data for sigma54 (69–198). PDB: 2K9M, BMRB: 15991

-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.[6] 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.[8]

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 initiation.

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.


[1] 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.
[2] 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. 53:367-376.
[3] 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.
[4] 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, 4129-4136.
[5] 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).
[6] Barrios, H., Valderrama, B., Morett, E. (1999) Compilation and analysis of sigma(54)-dependent promoter sequences. Nuc. Ac. res. 27, 4305-13.
[7] 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.
[8] Doucleff, M. et al. Structural basis of DNA recognition by the alternative sigma-factor, sigma54. Journal of molecular biology 369, 1070-8 (2007).
[9] 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.