Lecture 3: Sigma 54 Flashcards
How does the σ54 subunit function? How does it compare to σ70?
- σ54 has been implicated in directing the transcription of genes associated with nitrogen metabolism, various stresses and growth-limiting conditions including phage shock and pathogenicity.
- Much of the σ54 and σ70 are buried within the RNAP, except the DNA binding regions which are exposed on the surface.
- σ54 contacts -24 and -12 using 2 HTH known as RpoN and R1.
- There is no clear sequence homology between σ70 and σ54, however EM work suggest that they interact with the same area of the β and β’ subunits of RNAP.
- They also both have a region that interacts with RNAP, a region that inhibits transcription initiation and a region that binds specifically to the promoter DNA.
- Structural differences suggest that σ54 dissociates or relocates to allow RNAP to elongate, whereas σ70 can still loosely associate with the core during elongation.
- σ54 binds to a different sort of promoter (it has a K¬B), but it can’t directly melt the DNA (it has no k+2.
- In the initial RNAP-σ54 complex, the σ54 blocks the template DNA from entering the RNAP active site and the downstream DNA channel.
- σ54 therefore relies on transcription factors known as bacterial enhancer binding proteins (bEBPs) that interact with the DNA upstream of the promoter.
What are bacterial enhancer binding proteins? How are they structured?
- bEBPs have a regulatory domain, a DNA binding domain and a catalytic domain (which interacts with the σ54 RNA pol).
- bEBPs bind to an upstream activation sequence (UAS). It’s normally 80 to 150 bp upstream of the promoter.
- They form hexameric rings.
- The catalytic domain performs NTP hydrolysis.
How do bEBPs work? How do they interact with RNA pol? Draw a diagram.
- After binding to the UAS, the bEBPs cause DNA bending so they can interact with RNAP. This also involves the integration host factor (IHF).
- Then nucleotide hydrolysis induces structural changes in the closed complex and induce open complex formation.
- Cryo-EM indicates that the DB density Region I of the RNAP-σ54 holoenzyme is close to the -12. Region I prevents the initiation of transcription by obstructing the loading of DNA into the active-site channel of core RNAP. DB stands for bridging density. It is more pronounced in σ54, which is why the polymerase cannot spontaneously form an open complex.
- The bEBP activator causes the melting of DNA at the -12 position. It interacts with region I to relocate Db density. This results in the downstream movement of the DNA binding region of σ54, bringing the origin of the DNA melting near to the active site.
What are σ70-RNAP activators? How do they work?
σ70-RNAP promoters sometimes have a poor match to the -35 and -10 consensus sequences. In this case activators are required.
• The promoter of the lac operon contains non-consensus -35 and -10 regions. It requires CAP, catabolite activator protein (also known as CRP, cAMP receptor protein).
• To contact RNAP at the UP elements, -35 and -10, the DNA has to be distorted or bent.
• Opening/melting of the DNA at -10 then relieves this distortion.
• Activators like Crp/CAP improve K¬B by providing additional contacts for RNAP. They also improve k+2 by further distorting and bending the DNA.
What is the lac operon?
The lac operon is a model gene control mechanism which responds to sugar concentrations.
• An inducer such as lactose or allolactose will deactivate a repressor protein.
• Low or no glucose will lead to higher cAMP levels in cells. cAMP activates the CAP activator which will form a dimer and bind to DNA.
• When the preferred carbon source is present, permeases are inhibited. Catabolite repression also occurs.
• The lac operon produces β-galactosidase from lacZ and a permease from lacY.
How does CAP function? How is it structured and how does it bind to DNA?
- CAP is a homodimeric protein is activated by cAMP, which is produced in response to low levels of glucose.
- The N-terminal domain is involved in dimerization and cAMP binding (required for DNA binding and bending).
- C-terminal domain is involved in DNA binding through a helix-turn-helix motif.
- It binds to a site of about 22 bp at a responsive promoter. There is a 5 base pair consensus sequence. CAP binds most strongly to sites that contain two inverted versions of this pentamer.
- The pentamer can be adjacent to the promoter, upstream of the promoter or within the promoter itself.
- The AR1 site which interacts with the C-terminal domain of the α subunits of RNAP.
What are the three ways CAP can bind?
1) Class I: single site upstream of -35. Interaction with the α-CTD of RNAP via AR1 on a downstream CRP subunit. (E.g. lac).
2) Class II: single cap site replaces the -35 RNAP recognition region. AR1 interacts with the α-CTD and AR2 interacts with α-NTD. E.g. galP1.
3) Class III: multiple activators are involved. E.g. 2 or more CAP molecules or 1 CAP and 1 locus specific activator.
How did Zhou (1993) show the role of orientation in binding? Draw a diagram.
Experiments were used to show that different binding sites correspond to different orientations.
• Zhou (1993) created different heterodimers.
• One had a non-functional activating region but WT DNA binding specificity.
• The other had a functional activating region but non-WT DNA binding specific.
• This allowed researchers to orient the dimers.
• It was discovered that the promoter proximal subunit is involved in activation.
How do we know that activators function by promoter melting at -10? What can we use this for?
Use classical genetics to mutate DNA and screen for bacteria which induce lac operon in the presence of glucose and an inducer.
• LacUV5 promoter.
• Independent of CAP and cAMP.
• -10 sequence is different, it can be melted by σ70.
• LacUV5 is very useful because no activators are required, it is just controlled by repression.
What is FNR?
FNR is fumarate nitrate reduction and it is involved in oxygen deprivation response.
• FNR forms a dimer without oxygen and binds to DNA.
• Aspartate and arginine form part of the dimerization helix which forms salt bridges.
• 4Fe-4S is used sense oxygen presence.
• When oxygen is introduced the [4Fe-4S]2+ is converted to [2Fe-2S]2+.
• In aerobic cells further, oxygenation degrades the cluster to produce cluster less apo-FNR.
What are the two main FNR classes?
1) Class I: FNR binding is centred further upstream of the start site at around -61.5. FNR makes contacts with RNAP through AR1. Similar to CAP.
2) Class II: FNR binding is centred further upstream of the start site at -41.5. FNR makes contact through AR1, AR2 and AR3.
