Elongation complex stability
All RNA polymerases have about an 8 base pair RNA DNA hybrid in their elongation complexes; as a base is added at the 3′ end, an upstream base dissociates (resolves) from the hybrid and enters an RNA exit channel, before leaving the protein.
Why 8 base pairs?
RNA polymerases, from the small single subunit enzyme here, to much larger and unrelated RNA polymerases from bacteria and humans, all maintain an about 8 base pair RNA-DNA hybrid during elongation. Indeed, initial transcription is designed to stabilize the system until this length is achieved. Why 8 base pairs? It has been hypothesized that 8 base pairs is the length required to establish a topological locking of the RNA around the template strand DNA.
Note in the above that the red RNA strand, with its 3′ end in the active site (rightmost base) and forms an 8 bp RNA-DNA hybrid. The remainder of the 5′ end of the RNA exits the polymerase towards the “back” of this structure as shown. As such, the green nontemplate strand of the DNA and blue template strand cannot come together because their reannealing is blocked by the RNA. The transcription bubble is locked open. This is independent (and in addition to) any thermodynamic stability of the RNA-DNA hybrid (which is expected to be only somewhat more stable than the reannealed DNA-DNA duplex).
For this reason, elongation complexes that are stopped in the middle of a DNA stretch are stable for more than an hour. That is unless….
Bumping by a trailing RNA polymerase
Bumping by a trailing (lagging) RNA polymerase can displace a leading RNA polymerase (this is true for the T7 family, but not for the multi-subunit family of RNA polymerases). We have proposed that this happens by the trailing RNA polymerase “pushing” against the leading RNA polymerase, which can lead to (hyper) forward translocation (that is, without incorporating a base into the RNA). Hyper forward translocated complexes have removed the 3′ base (and the associated 3′ hydroxyl) from the active site and so are inactivated. Further hyper forward translocation reduces the size of the RNA-DNA hybrid and unthreads the topological lock. The resultant instability allows the complex to dissociate, ending transcription irreversibly.
How does an elongation complex come apart in the middle of the DNA, as occurs during a process called termination?
For this we need to “unthread” the topological locking of the RNA around the DNA. We believe this happens through a process called (hyper) forward translocation.
As you can see in the “bumping” cartoon above, a trailing RNA polymerase can “push” a stopped RNA polymerase, sliding the stopped polymerase forward. But in order for that to happen, the melted bubble must move in accordance (the structure of the complex requires this).
The cartoon above shows a single hyper forward translocation step (the bases are numbered arbitrarily to allow one to follow the movement). Note that the polymerase and the melted bubble move forward by one base. Movement of the bubble involves:
- melting of one base pair in the DNA (right-most red arrows)
- melting of one base pair in the RNA-DNA hybrid (leftmost red arrows)
- reannealing of one base pair upstream
The first two above are unfavorable, balanced only partly by the third favorable process. A “bumping” force from the left could be enough to drive this process.
This one base pair movement is probably not enough to unthread the lock (otherwise undesired termination might happen during normal elongation). But at some point, perhaps 4-5 hyper forward translocation events, the RNA may be sufficiently unthreaded to now be able to dissociate, allowing the bubble to collapse, releasing the polymerase.
Why doesn’t this happen more often? Perhaps it does, but not with enough forward translocation steps to reach the point of critical instability. Note that each hyper forward translocation event is reversible – the polymerase can reverse forward translocate.
The first hyper forward translocation event competes with the normal process of transcription elongation, as shown below.
The normal transcription cycle of repeated base addition and (normal) forward translocation is shown in the box above. Note that the very first hyper forward translocation event displaces the 3′ hydroxyl of the RNA from the active site; the polymerase cannot add another base until the complex reverse forward translocates back in register to re-position the 3′ hydroxyl.