A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs

Abstract

A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2'-deoxy, 2'-NH2 and 2'-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2'-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2'-substituents (2'-OMe and 2'-N3), and may also enhance use of NTPs with modifications at other than the 2'-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.

Figure 1

The templating base (cyan), 3′rNMP (red):template (blue) base pair and Tyr639 (yellow) and His784 (green) side chains as seen in the structure of a T7 RNAP initial transcription complex (PDB 1QLN) (7).

Figure 2

Incorporation of non-canonical NMPs with the wild-type, Y639F and Y639F/H784A enzymes. The NTPs (at 0.5 mM) present in each reaction are specified over each gel lane. (A) BglII cut pPK5 (9) as template. Lanes 1–4, wild-type; lanes 5–8, Y639F; lanes 9–12, Y639F/H784A. (B) BglII cut pPK5 as template. Lanes 1–4, wild-type; lanes 5–8, Y639F; lanes 9–12, Y639F/H784A. (C) HindIII cut pT75 (10) as template. Lanes 1–4, wild-type; lanes 5–8, Y639F; lanes 9–12, Y639F/H784A. (D) HindIII cut pT75 as template. Lanes 1–4, wild-type; lanes 5–8, Y639F; lanes 9–12, Y639F/H784A. The sequences of the transcripts obtained from the pPK5 and pT75 templates are presented to the left of the gels.