The architecture of RNA polymerase fidelity
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The architecture of RNA polymerase fidelity Craig D Kaplan* See research article http://www.biomedcentral.com/1741-7007/8/54
Abstract The basis for transcriptional fidelity by RNA polymerase is not understood, but the ‘trigger loop’, a conserved structural element that is rearranged in the presence of correct substrate nucleotides, is thought to be critical. A study just published in BMC Biology sheds new light on the ways in which the trigger loop may promote selection of correct nucleotide triphosphate substrates.
Replicative or transcribing nucleic acid polymerases must produce complementary copies of nucleic acid templates by a mechanism that strikes a fine balance between fidelity and speed. For many of these enzymes, this is achieved by high selectivity for the correct substrate, with a proofreading step for removing incorrect nucleotides if the selection step fails (for a recent review see [1]). A number of options exist for proofreading by polymerases. DNA polymerases recognize noncomplementary base pairs and translocate them to a different domain or subunit of the enzyme for excision. For multisubunit RNA polymerases (RNAPs), detection and correction of nucleotide misincorporation both occur in the same active site: incorrect nucleotides may be released before they have been incorporated; or the misincorporation may cause the enzyme to pause and undergo an active site reorganization that, sometimes with the participation of extrinsic cofactors, favors a nucleolytic removal of RNA containing misincorporated substrates. In broad terms, it is thought that binding of the correct complementary nucleotide to the DNA template in the RNAP active site induces closure of the site, with the correct alignment of critical amino acids for the polymerization reaction and thus efficient catalysis (see Figure 5 of [2]). The critical component in this structural rearrangement is the trigger loop, a flexible element of the largest subunit of RNA polymerase (the β’ subunit in *Correspondence: [email protected] Department of Biochemistry and Biophysics, Texas A&M University, TAMU 2128, College Station, TX 77843-2128, USA
eubacterial RNAP, and the Rpb1 subunit in eukaryotic RNA polymerase II (Pol II)) that interacts with the substrate and other elements of the enzyme active site. Removal of the trigger loop causes a drastic reduction in both the speed and the accuracy of nucleotide addition [3], which is consistent with the general picture sketched above; and substitution mutants within the trigger loop can either increase or decrease the RNAP elongation rate in vitro in Escherichia coli or Pol II (see for example [4]), suggesting selection for an optimum balance of speed with accuracy. The exact role of the trigger loop in selective binding and catalysis has, however, remained unclear. Recently, the understanding of the enzymatic activity of multisubunit RNAPs has reached a level of detail where the contributions of individual amino acid residues can be studied within an emerging structural framework, a
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