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Regulation from “remote” sites - 3 examples Repression in lac and other cases Activation via sigma54 Activation from a viral origin of replication. In a few cases repressors (and more rarely activators) bind outside their “zones.”. clues. Each typically contains another site within
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Regulation from “remote” sites - 3 examples Repression in lac and other cases Activation via sigma54 Activation from a viral origin of replication
In a few cases repressors (and more rarely activators) bind outside their “zones.” clues Each typically contains another site within the downstream zone. There is sometimes an IHF bending site in between. Some examples of sigma70 promoters with “far” upstream regulators act rep
act rep Here are examples of activators that work without a downstream partner They all use a different sigma, sigma54. (some of these also have IHF sites (open boxes).
In addition, when sigma70 RNA polymerase transcribes late T4 promoters, interactions at the replication origin activate transcription late viral promoters replication origin interactions here, needed for transcription here. (distances of kilobases)
Next set of questions. How does remote regulation work in the 3 systems repression of sigma70 transcription activation of sigma54 transcription activation of viral T4 transcription from ori
crp Polymerase -70 -50 Bgal gene promoter +1 leader O3 O1 O2 -90 +200 The lac repressor case is well understood Arrangement of lac regulatory elements O1 has the highest affinity for repressor, followed by O2 and then O3
Lac repressor Nat Struct Biol 2000 Mar;7(3):209-14 monomer Native tetramer
The repressor tetramer has 4 HTHs so it can bind 2 operators. (each operator is the usual inverted repeat) It will bind the 2 highest affinity sites, 01 and 02. What’s the function of 02? (old data on next slide) HTH HTH HTH HTH O2 O1
No repressor -remote No binding control less binding when remote site removed Binding changes pattern Footprint here Operator better protected when a remote operator is also present Remove this
Interpretation: Multiple operators increases the overall affinity of repressor for lac DNA. This means that higher occupancy of 01 blocks polymerase more effectively. (note that only O1 is in the repression zone) O2 O1 200 base pair DNA loop Subunit/site in the repression zone. This blocks RNAP function
Other repressors with remote sites are well known to assist binding to the sites within the repression zone by looping. Below, the araC repressor binding 2 sites remote repression site Transcription blocked here subunit/site targeted by DNA looping
crp Polymerase -70 -50 Bgal gene promoter +1 leader O3 O1 O2 -90 +200 Why are there remote sites? Probably to build in additional regulation when the upstream region is already crowded with required elements. Arrangement of lac regulatory elements O1 has the highest affinity for repressor, followed by O2 and then O3
Rare sigma70 remote activation - also via looping Some activators form multimers which requires the DNA to loop. Possible IHF binding site +1 -40 The presence of the downstream target subunit is critical to provide the contact to pol The upstream subunit can provide additional binding energy to bring the downstream subunit to the DNA. -40 pol +1
Sigma54-dependent remote activators: no downstream subunit Unusual characteristics of sigma54-dependent transcription: Sigma54 has no aa sequence similarity to other sigmas yet it binds the same core pol. ATP hydrolysis needed for transcription initiation (the ATPase activity is part of the activators) And of course it can be activated from remote sites without a downstream partner For a review see Buck et al -J Bacteriol 2000 Aug;182(15):4129-36 (updated later in this lecture)
Old experiments used DNase and permanganate footprinting: Before activation: Polymerase is at promoter (DNase) Activator is not (DNase) promoter DNA is closed (permanganate) After activation Polymerase is at promoter (DNase) Activator is at promoter (DNase) DNA is open (permanganate) An example of opening detected by permanganate attack on melted thymines
The activator loops to touch the polymerase (and then uses ATP to open the DNA).
Summary activator is signaled. key design point: Using a stable closed complex allows activation at a distance. This provides the downstream target for the looping activator. inactive target (already bound) , isomerizaton looping, then ATPase triggers opening Consider 2 questions: How does sigma54 hold melting in check to provide the target? How does activator overcome this?
The lack of binding to the top strand probe is not shown here recall: sigma70 melts by binding the top strand (core binds the bottom strand). Sigma54 fails to melt because it instead binds the bottom strand. Sigma70 prefers the top strand and this must be contacted to open the DNA. Guo y - Proc. Natl. Acad. Sci. USA, Vol. 98, 9020-9025, 2001
N Binds -12 Binds -24 C acidic Clues from genetics: Mutants that melt without ATP/activator can be isolated This suggests that the N-terminus is placed so as to block the normal promoter melting pathway. Blocking provides a target for a looping activator Activator then uses ATP to remove this N-terminal block. So, what’s known about the family of activators? Look at paper published a few weeks ago - next slides?
The pspF activator: a hexameric ring ATPase. It contains GAFTGA motifs (orange L1 loops) known to interact with sigma54. These (6) motifs line the central cavity. Rappas et al Science, Vol 307, Issue 5717, 1972-1975 , 25 March 2005
Trap a complex with sigma54 (by using the transition state analogue ADP-AlFx) Sigma54 appears to enter the central cavity of the activator. (not yet known if its via the N-terminus) sigma54 pspF ring These are cryo-EM reconstructions. Crystals could only be obtained on free pspF (no sigma, no analogue)
Compare to known structure of other activators. Primary difference is in location of (L1) GAFTGA motifs. (arrows represent change in position). Does this mimic changes that normally occur during activation? current model: 1. Sigma54 N-terminus blocks melting. 2. Activator loops to the targeted closed complex. 3. The activator cavity surrounds the N-terminus. 4. ATP re-models the N-terminus via L1 changes. 5. DNA opening and transcription occur.
Bacterial viruses: remote activation etc Consider 2 strategies, one used by T7 and one used by T4: T7: use bacterial sigma70 holo to produce a new polymerase that transcribes viral genes with high rate and specificity. Then take over cell. T4: hijack sigma70 holo and then alter it to transcribe viral genes. Then take over cell.
The T7 genome is injected (above). A few early genes have sigma70 elements and are transcribed by sigma70 pol. One is these is a single polypeptide T7-gene specific polymerase. It transcribes the phage genome, allowing completion of the life cycle.
This single 110 Kd pol transcribes faster and with higher specificity than coli holo. Recall that coli holo is 4x larger. This T7 polymerase reads the viral genes and that’s the end.
T4 is larger and more complicated These sigma70 early products modify pol so it can transcribeT4 middle genes better than bacterial genes. from Annu. Rev. Microbiol. 1998. 52:231-286 The modified polymerase prepares the virus for replication and late gene transcription. Late gene txn requires ongoing DNA replication.
The promoter sequences The most important factors
AsiA is a viral anti-sigma that inactivates sigma70 polymerase sigma asiA Mixture Gel filtration shows co-elution of asiA and sigma70 AsiA blocks transcription at sig70 promoters. (Other data shows it binds the transcription complex by engaging sigma region 4. JBC 272, 1997 pp. 27435-27443 update in EMBO J. 2004 Aug 4;23(15):2952-62.
Model for the middle gene transcription complex This complex transcribes only promoters containing motA elements because it has lost the usual -35 interaction. MotA and asiA are co-activators of middle txn. See J Mol Biol (2003) 326:679-690 for a slightly different model.
Now the phage needs to switch to late txn. Simple - gp55 is a middle gene product. It can replace sigma70 and transcribe late genes. Complex - other middle proteins are needed. These are T4 replication proteins. In fact, late txn. stops if replication stops. The next sketches summarize conclusions primarily from Cell 1994 77:225-37 and Science (1992) 256:1298 (Peter Guiduschek lab)
Looping? tracking/sliding by replication machinery?
Looping as usual? Usual tracking/sliding by replication machinery?
tracking rationale - gp45 is part of a family of sliding clamps
model based on current data - the clamp asssociates by being sandwiched between RNAP-bound gp55 and gp33. from Geiduschek - Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17365-70. late “basal” transcription late replication-activated transcription provides upstream anchor questions remain: What parts travel with the clamp? Clash of transcription and replication? Does clamp direct RNAP binding or isomerization?
Homework: Suggest a detailed molecular model for how ATP is used to activate sigma54-dependent transcription. (Be sure to use lecture material as part of your answer.)