A New Model For Elongation Complex Stability in RNA Polymerase - the “topological Lock”

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What gives rise to the characteristic stability of an elongating (but not initiating) RNA polymerase and why do all RNA polymerases possess an 8-10 bp RNA:DNA hybrid? Conventional thinking posits that this length of duplex, together with protein-nucleic acid interactions, yields the required thermodynamic stability. We have proposed instead that the wrapping of 8-10 bp of RNA around the template DNA provides a topological locking of the RNA into the complex, preventing collapse of the DNA bubble. We have previously demonstrated that complexes dissociate primarily from “forward translocated” states, in which forward movement of the complex without incorporation of nucleotides leads to a shortening of the hybrid, but also to an unthreading of the lock, allowing dissociation. In complexes halted at the end of a homopolymeric stretch of T in the template DNA, the complementary RNA can slip back a base, reexposing a templating T, and allowing the incorporation of an additional A into the RNA. This slippage process repeats to generate a very long poly(A) tail. Our results show that upon depletion or removal of ATP, the RNA slips diffusively such that both the 3′ and 5′ ends of RNA extend out of the protein. Consistent with predictions of the topological lock model (but not of the thermodynamic model), these complexes are more stable than conventionally halted elongation complexes. We have further prepared halted complexes with 4 or 0 (zero!) hybrid base pairs and these complexes are also exceptionally stable, arguing that topological locking of the RNA, rather than thermodynamic stability, prevents complex dissociation. This has implications for mechanisms of termination and explains why phage, bacterial, and eukaryotic RNA polymerases all contain an 8-10 base pair hybrid, despite the very different sizes of the proteins.