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.

INTRODUCTION

The robust activity and strict promoter specificity of the single-subunit phage RNA polymerases (RNAPs) makes them ideal for synthesizing specific RNAs in vitro (1). Mutation of phage T7 RNAP Tyr639 to Phe eliminates discrimination of the hydrogen-bonding potential of the 2′-substituent of the substrate NTP (2). This mutant enzyme can be used to incorporate 2′-dNMPs, 2′-F-NMPs and 2′-amino-NMPs into transcripts (3) to make them RNase resistant (4), for studies of RNA structure–function relationships (5) or to expand the chemical repertoire of ribozymes.

However, this mutant is not as useful for incorporating NMPs with bulkier substituents (2′-OMe groups) into transcripts (6). The barrier to utilizing such substrates may not be in incorporating them into the RNA, but in subsequently extending the transcripts, i.e. many non-canonical NMPs may act as effective chain terminators. The Y639F mutation does not overcome this barrier because the tyrosine is positioned so as to discriminate the structure of the substrate NTP (Fig. 1) (7), but is not involved in sensing the structure of the 3′-NMP of the RNA. The crystal structure of a T7 RNAP transcription complex (7) reveals that the side chain of His784 occupies the minor groove side of the 3′-rNMP:template base pair (Fig. 1). Though this histidine is well conserved in the phage RNAPs, an H784A mutant has near wild-type activity but exhibits enhanced extension of RNAs with mispaired 3′-termini (8). These observations suggest that the H784A mutation might relax the barrier to extension of transcripts containing non-canonical NMPs at the 3′-end of the RNA. In particular, replacement of the bulky histidine side chain with alanine might make room for extending RNAs with 3′-NMPs carrying bulky minor groove substituents.

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).

MATERIALS AND METHODS

RNAPs were prepared as described (8). Transcription reactions were carried out for 30 min at room temperature in 40 mM Tris–HCl pH 8.0, 6 mM MgCl₂, 10 mM NaCl and 2 mM spermidine, with templates at 10^–7 M and RNAPs at 2 × 10^–7 M. All NTPs were used at 0.5 mM, and transcripts were labeled by inclusion of 1% (v/v) 800 Ci/mM [α-³²P]GTP in the reaction. Modified NTPs were from Trilink Biotechnologies. Reactions were stopped by addition of an equal volume of 90% formamide, 50 mM EDTA and 0.01% xylene cyanol, resolved by electrophoresis in 20% acrylamide, 1% bisacrylamide, 1× TBE gels, and visualized on a Molecular Dynamics phosphorimager.

RESULTS

We tested the ability of the wild-type and the Y639F and Y639F/H784A mutants to utilize 2′-OMe-UTP, 2′-OMe-CTP, 2′-azidoUTP and 2′-azidoCTP in RNA synthesis. On a template [BglII cut pPK5 (9)] in which CTP and UTP are first incorporated at +14 and +15, respectively, the wild-type enzyme can synthesize 34 nt run-off transcripts in reactions in which UTP is replaced with 2′-OMeUTP (Fig. 2A, lane 2) or in reactions in which CTP is replaced with 2′-OMeCTP (Fig. 2A, lane 3). Relative to the reaction with four NTPs (Fig. 2A, lane 1), yields of run-off transcripts are reduced 6- and 21-fold in the 2′-OMeUTP and 2′-OMeCTP reactions, respectively. When both UTP and CTP are replaced with the corresponding 2′-OMeNTPs, run-off transcription with the wild-type enzyme is undetectable (Fig. 2A, lane 4). Terminated transcripts are seen in these reactions at positions where 2′-OMeUMP or 2′-OMeCMP are incorporated (Fig. 2A, lanes 2–4). The Y639F mutation enhances utilization of the 2′-OMeNTPs: relative to reactions with four NTPs (Fig. 2A, lane 5), run-off transcription with Y639F is reduced, respectively, 2-, 8- and 55-fold in reactions with 2′-OMeUTP or 2′-OMeCTP or both (Fig. 2A, lanes 6–8). With the Y639F/H784A double mutant the reduction in run-off transcript synthesis is <15% in reactions with 2′-OMeUTP (Fig. 2A, lane 10) and only 3- and 6-fold in reactions with, respectively, 2′-OMeCTP or both 2′-OMeUTP and 2′-OMeCTP (Fig. 2A, lanes 11 and 12; data on the relative activity of the mutant enzymes with non-canonical NTPs is summarized in Table 1).

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.

Table 1. Activity of mutant RNAPs in reactions with 2′-OMe- or 2′-azidoNTPs.

	Wild-type	Y639F	Y639F/H784A
Template: BglII cut pPK5
4 rNTPs	100	100	100
2′-OmeU	17	50	90
2′-OmeC	5	13	35
2′OMeC+2′OmeU	n.d.a	2	17
2′-AzU	6	30	60
2′-AzC	2	30	60
2′AzU+2′AzC	n.d.	9	35
Template: HindII-cut pT75
4 rNTPs	100	100	100
2′-OmeU	9	20	60
2′-OmeC	n.d.	n.d.	0.14
2′-AzU	6	12	60
2′-AzC	n.d.	1.5	14
2′AzU+2′AzC	n.d.	1.5	14

‘Activity’ reflects synthesis of full-length run-off transcripts and is normalized to that seen in reactions with four rNTPs which is assigned a value of 100 in arbitrary units.

^an.d., not detectable.

Identical experiments were carried out with 2′-azidoUTP or 2′-azidoCTP replacing UTP or CTP, respectively (Fig. 2B). With the wild-type enzyme, run-off transcription was reduced 15- and 50-fold in reactions with 2′-azidoUTP or 2′-azidoCTP, respectively (Fig. 2B, lanes 2 and 3), and was undetectable in reactions with both 2′-azidoUTP and 2′-azidoCTP (Fig. 2B, lane 4). With Y639F, use of 2′-azidoUTP or 2′-azidoCTP reduced run-off transcription by 70% (Fig. 2B, lanes 6 and 7) compared to reactions with four NTPs (Fig. 2B, lane 5), while use of both 2′-azidoUTP and 2′-azidoCTP reduced run-off transcription by 11-fold (Fig. 2B, lane 8). With Y639F/H784A use of a single azido-modified NTP reduced run-off transcription by only 40% (Fig. 2B, lanes 10 and 11), while use of two 2′-azidoNTPs reduced it by 3-fold (Fig. 2B, lane 12).

With the pPK5 template UMP and CMP are not incorporated into the first 9 nt of RNA. Incorporation of non-canonical NMPs during synthesis of the first 9 nt of RNA is expected to cause greater reductions in run-off transcript synthesis because the RNA is readily released from the transcription complex during this initial phase of transcription. Therefore, if incorporation of a non-canonical NMP is slow, the RNA will usually dissociate before the NMP is incorporated. Once the RNA is >9 nt in length the transcription complex becomes much more stable, and is unlikely to release the RNA during the time required for incorporation of a non-canonical NMP. To see whether NMPs with bulky 2′-substituents could be incorporated during the initial phase of transcription we used HindIII linearized pT75 (10). On this template, UMP is first incorporated at +14 while CMP is incorporated at +6 and +7. In reactions with 2′-OMeUTP run-off transcript synthesis is reduced 11-, 5- and <2-fold with the wild-type and Y639F and Y639F/H784A mutants, respectively (Fig. 2C, lanes 2, 6 and 10). However, in reactions with 2′-OMeCTP run-off transcription is undetectable with the wild-type or Y639F enzymes (Fig. 2C, lanes 3, 4, 7 and 8), and is reduced ∼700-fold with Y639F/H784A (Fig. 2C, lanes 11 and 12).

Run-off transcript yields in reactions with HindIII cut pT75 and 2′-azidoUTP are reduced 18-, 8.5- and <2-fold with the wild-type and Y639F and Y639F/H784 mutants, respectively (Fig. 2D, lanes 2, 6 and 10), relative to reactions with four NTPs (Fig. 2D, lanes 1, 5 and 9). In reactions with 2′-azidoCTP or both 2′-azidoCTP and 2′-azidoUTP, run-off transcription is undetectable with the wild-type enzyme (Fig. 2D, lanes 3 and 4), is reduced 65-fold with Y639F (Fig. 2D, lanes 7 and 8), but is reduced only 7-fold with Y639F/H784A (Fig. 2D, lanes 11 and 12).

DISCUSSION

By introducing an H784A substitution into a Y639F background, we obtain a polymerase with an enhanced ability to incorporate NMPs with bulky 2′-substituents into RNA. In reactions with 2′-OMe- or 2′-azido-modified NTPs yields of run-off transcripts, relative to reactions with the four canonical NTPs, are markedly increased with the double mutant and premature termination products are greatly reduced or eliminated. Incorporation of the modified NMP is more efficient if it occurs during elongation, rather than during the poorly processive initiation phase of transcription when the RNA is <9 nt in length. However, the double mutant does incorporate azido-NMPs efficiently, even during initial transcription (Fig. 2D, lanes 11 and 12). Given the position of the His784 side chain in the crystal structure of an initial transcript complex (7), it is likely that the effect of the H784A mutation is due primarily to enhanced extension of RNAs containing a modified NMP at the 3′-terminus. This would be consistent with the observation that the H784A mutation enhances extension of RNAs with mispaired 3′-termini, but does not increase incorporation of mispaired NMPs (8). This also suggests that the H784A mutant, either alone or in a Y639F background, might be useful in synthesizing RNAs with NTPs containing modifications at other than the 2′-position. In this study we tested 2′-OMe-NTPs and 2′-azidoNTPs because these are commercially available and are not readily used by either the wild-type or Y639F polymerase. Evaluation of the utility of the H784A and Y639F/H784A mutants in synthesis with differently modified NTPs is probably best done on a case-by-case basis.