Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription

Abstract

We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10(3) over the error rate determined solely by selectivity (1.8 x 10(-4)). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.

Fig. 1.

A Pol III form devoid of RNA cleavage activity incorporates a mispaired nucleotide. (A) Template-strand sequence in the beginning of SUP4 (gray) and the sequence of the 17-mer RNA (black). (B) (Upper) Shows the relevant sequence of SUP4 (gray) and the 3′-terminal sequence of the 17-mer (black), with X denoting the position at which the NTP incorporation was tested. (Lower) Purified ternary complexes containing radiolabeled 17-mer SUP4, Pol III, or Pol IIIΔ were analyzed either directly (lanes 1 and 6) or after a 5-min incubation with 600 μM of the noted NTP. Arrow, residual 18-mer; asterisks, slippage product. (C) SUP4 was transcribed for 5 min in the presence of ATP, CTP and radiolabeled UTP by Pol III, Pol IIIΔ alone, or Pol IIIΔ preincubated for 10 min with the indicated purified recombinant subunits.

Fig. 2.

Pol III selectivity determined in the absence of RNA-cleavage activity. (A) (Upper) Shows the relevant sequence of SUP4 (gray) and the 3′-terminal sequence of the 17-mer (black) with the newly added bases in bold. (Lower) Time-course analysis of incorporation of GTP (0.6 μM) by ternary complexes containing SUP4, Pol IIIΔ and the radiolabeled 17-mer. For each reaction time, the amount of the residual 17-mer was quantified and expressed as percentage of the initial amount (t = 0) after subtraction of the lowest quantity of 17-mer remaining unreacted. Data were fitted to a single-exponential function and the apparent rate constant (k_a) calculated. (B) Same as in A, except that the ternary complexes were incubated with ATP (600 μM) instead of GTP. The mismatch formed is framed on the sequence representation. (C) Same as in A except that the ternary complexes were preincubated for 10 min with the cleavage-incompetent rC11^E92H and r(C37-C53), before being allowed to react with either GTP (0.6 μM) (Left) or ATP (600 μM) (Right).

Fig. 3.

Time-course analysis of RNA cleavage by Pol III. (A) Time-course of RNA cleavage in transcription buffer in the absence of NTPs by purified ternary complexes containing SUP4, Pol III, and the radiolabeled 17-mer. Data were analyzed as described for Fig. 2A. (B) Purified ternary complexes containing SUP4, PolIIIΔ and either the (radiolabeled) correctly 3′-end-paired 17-mer or 3′-end mispaired 18-mer transcript were preincubated for 10 min with wild-type rC11 and r(C37–C53) and the RNA-cleavage reaction was initiated by starting incubation in cleavage buffer (at 16°C). The data were analyzed as described for Fig. 2A.

Fig. 4.

Addition of a correct nucleotide after misincorporation is extremely inefficient. (A) (Upper) The relevant sequence of SUP4 (gray) and the 3′-terminal sequence of the 18-mer (black) with the newly added bases in bold and the mismatches framed. (Lower) The time-course analysis of GTP (600 μM) incorporation by purified ternary complexes containing SUP4, Pol IIIΔ, and the radiolabeled 3′-end mispaired 18-mer transcript. (B) The data were analyzed as described for Fig. 2A.

Fig. 5.

Modeling the reactions determining Pol III fidelity. Diagram describing the reactions determining the fidelity of Pol III transcription, where: (n − 1) − RNA species of length (n − 1) within a Pol III ternary complex, (n) − product of the correct incorporation at the template position n, (n*) − product of misincorporation at position n, (n + 1) − product of the extension of the correctly paired species (n), and (n* + 1) − product of the extension of the incorrectly paired species (n*).