8.1 - Transcription Flashcards
How is transcription carried out in eubacteria?
By RNA polymerases and sigma factors that guide it to specific promoters
What is a core RNA polymerase?
An RNA polymerase enzyme without the sigma factor
What does the core RNA polymerase enzyme consist of?
2 alpha (2α), 1 beta (β), 1 beta prime (β’) and 1 omega (ω) subunit
What does the C-terminus of the alpha subunit interact with and why?
Regulators because it is flexible and can therefore reach out.
What is a feature of the core enzyme?
It itself does not recognize any specific DNA sequence and thus has the ability to transcribe RNA from non-specific initiation sequences.
How can we get the core enzyme to intiate transcription at specific promoters?
With the addition of a sigma factor
What regions does the sigma factor bind to on promoters?
They recognize and bind to specific sequences on the promoter, these are the -35 and -10 regions.
What characteristic allows sigma factors to recognize and bind DNA?
The fact that they are elongated
What happens to the sigma factor once transcription starts?
It is ejected from the holoenzyme
What makes a promoter strong?
Strong promoters have an A/T rich regions that will be recognized by the alpha subunit
What can RNA polymerases also do in the case of strong promoters?
The C-terminus of the alpha subunit is also involved in sequence recognition in the case of very strong promoters.
What is an example of a strong promoter?
The promoter of rRNA
Name some features of RNA polymerase
(1) Proteins which bind TATA box
(2) Kinases to phosphorylate subunits
What are the beta prime subunits for in bacterial RNA polymerase?
They are catalytic subunits and are where the RNA is synthesized
How is transcription initiated?
RNA polymerase does random walk till it comes to strong sequence it recognizes (promoter). There is binding affinity and when docked on promoter it needs to unwind DNA and whole polymerase needs to go through conformational change. This requires activation energy. Reaction goes forward due to phosphates that are hydrolyzed. This happens at certain rate because you have to overcome certain activation energy
What is the intrinsic promoter strength?
A reaction between RNA polymerase, its sigma factor and DNA. It does not take into consideration other regulating factors.
What can the intrinsic promoter strength be broken down into?
(1) Strength of binding
(2) Strength of unwinding DNA
What is strength of binding?
How strongly does RNA pol bind DNA. This is expressed by a binding constant (Kb) which is an equilibrium constant meaning that the reaction can go back and forth (binding and unbinding
What is strength of unwinding DNA?
How efficiently can RNA pol I unwind DNA at the promoter to initiate transcription. This is expressed by a rate constant called isomerization constant (Kf). Kf is not an equilibrium constant because the reaction doesn’t go back and forth
What does a strong promoter have in terms of binding and unwinding?
Weak binding constant (weak Kb) but a strong isomerization constant (strong Kf).
What do the strongest promoters have?
Both strong Kb and strong Kf.
What influences the isomeration constant (Kf)?
The sequence of the TATA box.
What is the formula for promoter strength?
promoter strength = binding x opening rate constant
What does RNA polymerase not have when it enters the elongation step?
Sigma factor because it is ejected from the holoenzyme once transcription starts
What are the 2 elongation factors that bind to RNA polymerase?
(1) NusA
(2) NusG
What does NusG do?
NusG forms a ring around the DNA that helps the RNA polymerase to continue transcribing without falling off; so like a processivity-promoting factor (analogous to clamp in DNA replication).
What does NusA do?
NusA will help eliminate secondary structures of RNA during transcription.
Where does RNA polymerase stop transcribing?
At sequences called transcription terminators
What are the two termination mechanisms in E.coli and most bacteria?
(1) Rho dependent termination
(2) Rho independent termination
What does the Rho protein bind to?
A specific RNA sequence called transcription termination pause site
What are transcription termination pause sites?
A G-C rich region on the RNA that is being synthesized
What happens when Rho recognizes and binds to the termination pause site?
It will use energy from ATP hydrolysis to translocate along the RNA until it reaches the DNA-RNA helical region (3’ end of the RNA being synthesized). - On its way to the DNA-RNA helical region it will unwind the RNA from the DNA template.
What family of proteins does Rho belong to?
Rho is a RNA-DNA helicase (uses ATP), it is part of the AAA+ family.
What causes termination in rho dependent termination?
The contact between RNA polymerase and Rho
How is a stem loop structure formed in rho independent termination?
The mRNA contains a G-C rich sequence that can base pair with itself to form a stem-loop structure (due to inverted repeats region) of 7-20bp in length.
What is an important properties of G-C bonds?
C-G have significant base-stacking interactions and can form 3 hydrogen bonds between each other, resulting in a stable RNA duplex.
What are base stacking interactions?
Interactions between adjacent base – pairs, very important in stability.
What follows the stem loop structure in rho independent termination?
There is a sequence of uracil residues. Bonds between uracil and adenine are very weak. NusA protein, which is bound to RNA polymerase, will bind to the G-C stem loop tightly enough causing the RNA polymerase to stall when transcribing the Uracil rich region following the stem loop.
What do the weak adenine-uracil bonds do?
They will lower the energy of destabilization required for the RNA-DNA duplex unwinding. As a result, RNA will unwind from the template and dissociate from the RNA polymerase
