Abstract
We suggest a novel two-step proofreading mechanism with two sequential rounds of proofreading selection in mRNA transcription. It is based on the previous experimental observations that the proofreading RNA polymerase cleaves off transcript fragments of at least 2 nt and that transcript elongation after a nucleotide misincorporation is anomalously slow. Taking these results into account, we extend the description of the accuracy of template guided nucleotide selection beyond previous models of RNA polymerase-dependent DNA transcription. The model derives the accuracy of initial and proofreading base selection from experimentally estimated nearest-neighbor parameters. It is also used to estimate the small accuracy enhancement of polymerase revisiting of previous positions following transcript cleavage.
Highlights
The accuracy of an enzymatic reaction reflects the enzyme’s preference of a correct substrate over other substrates in product formation
The free energy difference, ΔG‡,nc - ΔG‡,c, between the standard free energy of template binding for a cognate and a non-cognate nucleotide in the catalytic site of the polymerase determines the accuracy of substrate selection
The flawed and the following base pair will both be cleaved off (Figure 3). From these considerations we propose that the accuracy of template dependent nucleotide selection by RNA polymerase is maintained by one initial selection step and two proofreading steps (Figure 3)
Summary
The accuracy of an enzymatic reaction reflects the enzyme’s preference of a correct substrate over other substrates in product formation. Genetic information transmitting reaction systems like transcription, translation and replication require high accuracy, since a flawless product from any of these processes depends on a chain of multiple elongations events, all of which must be correct. A further challenge in transcription is that the same polymerase structure must probe several types of substrates, which are correct or incorrect only depending on the DNA template in the active site. The free energy difference, ΔG‡,nc - ΔG‡,c, between the standard free energy of template binding for a cognate and a non-cognate nucleotide in the catalytic site of the polymerase determines the accuracy of substrate selection. Precise experimental estimates of transcriptional accuracy and the contributions from initial and proofreading selection have remained hard to come by [2,9]
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