Stepwise mechanism for transcription fidelity

Abstract

Background: Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.

Results: Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH.

Conclusions: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

Figure 1

Folding of the Trigger loop (TL) restructures an amino acid content of the active centre. The active centre of the Thermus thermophilus RNAP elongation complex with bound substrate is shown (there is complete conservation of sequence in TL and active site residues between T. thermophilus and T. aquaticus (used in this study) RNAPs). Unfolded TL (magenta) and folded TL (green; PDB 2PPB and 2O5J, respectively [9]) in the active centre are shown as ribbons. The amino acids of the TL that face the nucleotide triphosphate (NTP; orange) bound in the i+1 site upon TL folding, and that were analysed in our study, are shown as coloured sticks (Q1235 grey, M1238 yellow, R1239 blue, F1241 green, H1242 cyan, T1243 red) in both folded and unfolded states of TL. The NTP in i+1 site is orange, RNA is red and template DNA in grey. Mg²⁺ ions of the active centre are shown as spheres.

Figure 2

Incorporation and misincorporation by wild-type (WT) and mutant RNA polymerase (RNAPs). (a) Cartoon schematically describes the reaction of cNTP (cGTP) incorporation in EC^G1 (Additional File1: Figure S2) with ³²P 5'-labelled RNA (asterisk). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Representative gels of 100 μM cGTP incorporation by WT and 500 μM cGTP incorporation by ΔTL in EC^G1 are shown. The lack of complete extension of transcripts was due to the procedure by which elongation complexes were assembled (see Methods). (b) The intrinsic proofreading reaction accompanies misincorporation. The cartoon above the gel schematically describes the processes going on during ncGTP misincorporation in EC^A (Additional File 1: Figure S2). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Note that RNAs in elongation complexes were labelled at the 3' end (by incorporation of [α³²P]GTP; asterisk), thus allowing monitoring both misincorporation event and removal of the wrong nucleotide via transcript assisted proofreading. Misincorporation of 1 mM ncGTP and proofreading by WT, H1242A and R1239A RNAPs are shown as an example. The cleavage products larger than dinucleotide originate from 2 bp and 3 bp backtracked complexes that undergone further extension after misincorporation. The colours of the RNA products of the reactions are the same as in the scheme of the reaction above the gels. Black vertical line separates lanes originating from the same gel that were brought together.

Figure 3

Roles of TL amino acids in catalysis. (a). Reaction rates in saturating (1 mM) cGTP in EC^G1 and K_d [ncGTP] by wild-type (WT), M1238A, M1238V and M1238L are shown below the cartoons of the active centres of the corresponding enzymes, drawn in PyMol using PDB 2O5J and 'mutagenesis' function (colour code as in Figure 1). (b) Kinetics of pyrophosphorolysis by WT, R1239A and H1242A in the presence of 0.5 mM PP_i in EC^G1 (Additional File 1: Figure S2) with ³²P 5'-labelled RNA that was walked by two positions (G and A). (c) Kinetics of intrinsic transcript hydrolysis by WT (red squares), Q1235A (violet circles) and R1239A (green circles) RNA polymerase (RNAP) in EC^hydr (Additional File 1: Figure S2) with ³²P 5'-labelled RNA. The lines in the plot are the non-linear regression fits of the data. (d) pH dependences of the rates of 1 mM cGTP incorporation in EC^G1 at 20°C by WT, R1239A, H1242A and R1239A/H1239A RNAPs. Solid lines show fits of the data to a sigmoidal function, and pK_a values retrieved from these fits are shown above the plots. (e) Kinetics of 1 μM cGTP incorporation in EC^G1 by WT (red squares), R1239A (green circles) and R1239N (blue triangles). Solid lines show fits of the data to an exponential function.

Figure 4

Role of Q1235 in discrimination against c2'dNTPs and c3'dNTPs and in catalysis. (a) Kinetics of incorporation of saturating cGTP (1 mM) in EC^G1, c2'dATP (4 mM) and c3'dATP (1 mM) in EC^A by wild-type (WT; red squares) and Q1235A (violet circles) RNAPs. Kinetic discrimination against c2'dATP and c3'dATP was quantified as a ratio of the rate of cGTP incorporation to the rate of incorporation of corresponding erroneous substrate. (b) Q1235 does not participate in catalysis directly. Kinetics of saturating (1 mM) cGTP incorporation in EC^G1 by R1239A/H1242A (orange circles) and Q1235A/R1239A/H1242A (cyan triangles) RNA polymerase. Compare to panel A (left plot) and to Figure 3e.

Figure 5

The stepwise mechanism of transcription fidelity. Cartoon schematically shows RNA polymerase active centre and the steps of discrimination against various erroneous substrates. From top to bottom: incorporation of complementary nucleotide triphosphate (cNTP), misincorporation of ncNTP, c2'dNTP and c3'dNTP. Discrimination against unNTP (unusable substrates) takes place in the open state of the active centre and is not shown. The approximate discrimination contributions, found in our study, are shown for each step.