Structural and mechanistic studies of transcription activation
Transcription is a fundamental process of life, allowing organisms to utilise the information stored in DNA to respond to the changing environment. Transcription is therefore tightly controlled by regulator proteins, which allow genes to be turned on and off in order for cells to develop, differentiate and adapt. Regulators are often the final players in complex signalling pathways.
In order to express the right gene at the right time, it is vital to ensure that transcription is specific to the required genes at a given time. In bacteria, this is achieved using σ factors, proteins that bind to the multi-subunit RNA polymerase and recruit it to the promoter DNA at the start of the target gene. When it binds to the promoter, the RNAP-σ complex melts the DNA to initiate transcription.
In the lab we study the central enzyme of transcription, RNA polymerase, and its interactions with cofactors and activators using a range of structural and biochemical techniques. Understanding the mechanisms of transcription regulation not only offers insights into a fundamental biological process, but also can provide avenues to explore novel antibiotics. We are especially interested in the bacterial transcription machinery, which represents a simplified model system for studying transcription activation in all organisms. This project is in collaboration with Prof. Martin Buck.
Figure 1: σ54 RNAP forms a closed complex with promoter DNA which is unable to initiate transcription. The closed complex is isomerised by σ54 activators using energy released from ATP hydrolysis, allowing DNA melting and loading of the template DNA strand into the active site to form an open complex.
There are two classes of bacterial σ factor, σ70 and the less common σ54. σ54 controls transcription from a diverse range of genes important for cell survival, including those involved in stress responses, virulence, adaptation and nitrogen metabolism. The mechanism of σ54 transcription activation is distinct from the σ70 class and conceptually similar to that of eukaryotic transcription. σ54–RNAP is unable to initiate transcription alone, instead forming a closed complex with the promoter DNA. This closed complex is the target for σ54-activator proteins, members of the AAA+ family of ATPases that use energy released from ATP hydrolysis to remodel their substrate. σ54-activators are specific to particular promoters, so that σ54 can activate transcription from a wide range of promoters independently.
σ54-activators bind specifically to sequences located upstream of the promoter, sometimes by as much as 150bp, and contact the closed complex by ‘looping out’ the DNA in between. They then use the energy released from ATP hydrolysis to isomerise the closed promoter complex, causing DNA melting and initiating transcription. The mechanism of σ54 therefore provides a simplified model system to capture the very earliest stages of transcription activation, involving the melting out of double stranded DNA and the delivery of the DNA template strand into the active site of RNAP.
Structural studies of PspF and its interaction with sigma54
We are using the σ54 activator Phage shock protein F (PspF) to study the structural basis for the remodelling of the σ54 closed promoter complex by σ54 activators during transcription activation. Using a combination of x-ray crystallography and single particle cryo-electron microscopy in conjunction with mutational data, we are observing how changes within the active site of PspF are relayed to σ54.
Figure 2: Using a combination of cryo-EM and X-ray crystallography, we showed that σ54 activators contact σ54 using two surface-exposed loops (L1 and L2). These undergo ATP hydrolysis-dependent movements due to rotations of helix 3 and form a stable interaction with σ54 at the point of ATP hydrolysis (modelled using the ATP hydrolysis transition state analogue ADP.AlFx).
We have solved the crystal structure of the PspF1-275 AAA+ domain at 1.75 Å and the structure of the PspF/σ54 complex using cryo-EM. Together, these results have enabled us to propose a mechanism for the process of energy transfer (energy coupling) from PspF to the closed promoter complex. For more information, please refer to Rappas et al., 2005 and download the explanatory movies.
Figure 3: High-resolution X-ray crystallographic structures of the σ54 activator PspF bound to various nucleotides and nucleotide analogues. A conformational signalling pathway links the active site to the L1 and L2 loops. Nucleotide hydrolysis triggers a 'Glutamate switch' (A), which drives conformational changes through the signalling pathway that result in movements of the two loops (B).
We have determined high-resolution structures of PspF in complex with different nucleotides and nucleotide analogues. Our structures highlighted a switch pair (Glu-Asp) that senses the nucleotide state within the active site of σ54 activators during the ATP hydrolysis cycle. The switch transmits the changes through a conformational signalling pathway to the σ54 interacting site of PspF. For more information, please refer to Rappas et al., 2006.
Snapshots of transcription activation
Why does the RNAP-σ54 form a closed complex that is unable to proceed to open complex and leads to transcription? Our recent study using cryo-electron microscopy and single particle reconstruction, combined with labelling techniques, provided a structural explanation. The -12 promoter DNA, where double stranded DNA starts to melt to form the transcription bubble, is located too far upstream relative to the RNAP active site for the melted template DNA to enter the active site. Furthermore, regions of σ54 block the entrance of the melted template strand DNA into the active site. Our structure of the activator bound RNAP-σ54 complex reveals that σ54 activators interact with the region where the physical blockage occurs, destabilising and eventually removing the blockage. The activator also produces domain movements within the σ54 –RNAP that cause the promoter DNA to slide in a downstream direction, allowing the correct positioning of the melted template strand relative to the RNAP active site. For more information, please refer to Bose et al., 2008 and download the explanatory movies.
Figure 4: Cryo-EM studies of σ54, RNA polymerase and PspF. σ54–RNAP (A) and σ54 RNAP bound to PspF at the point of ATP hydrolysis (B and C; modelled using the ATP hydrolysis transition state analogue ADP.AlFx). Panels B and C show that the activator causes large-scale rearrangements of the σ54 domains. These serve to remove a block to DNA loading and reposition the promoter DNA in a location where the template strand can access the active site of the RNAP.
Mechanism of AAA+ proteins
We are also interested in the mechanism of a number of AAA+ proteins and in general how AAA+ proteins convert chemical energy into mechanical forces. AAA+ proteins are relatively poor ATPases in comparison to metabolic ATPases, and their ATPase activity is often significantly regulated by binding to their substrate ligand. In collaboration with Dr Dale Wigley at Cancer Research UK, we analysed a large set of AAA+ crystal structures and identified a conserved link between the ATPase activity of AAA+ proteins and ligand binding. We show that the Glutamate switch pair, which we initially identified in PspF, could act as a switch to convert the AAA+ proteins from an inactive to an active conformation in response to ligand binding. For more information, please refer to Zhang and Wigley, 2008.
Figure 5: Conservation of the glutamate switch among AAA+ proteins. Panel a: Sequence alignment highlighting the conservation of the glutamate and asparagine residues that form the switch among AAA+ proteins. Panel b: Plot of side chain torsional angles reveals that the switch glutamate commonly occupies either of two conformations in the available crystal structures, representing an active and an inactive switch conformation.
Regulation of σ54 activators
We are also studying the mechanisms by which the activities of two transcriptional activators, NorR and PspF, are activated in order to turn expression from σ54 dependent promoters on and off.