Nucleic Acids Research, 2002, Vol. 30, No. 24 e138
© 2002 Oxford University Press
A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs
Robert Padilla and
Rui Sousa*
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
*To whom correspondence should be addressed. Tel: +1 210 567 8782; Fax: +1 210 567 8778; Email: sousa{at}biochem.uthscsa.edu
Received August 14, 2002; Revised October 4, 2002; Accepted October 20, 2002
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ABSTRACT
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A T7 RNA polymerase in which Tyr639 is mutated to Phe readily
utilizes 2'-deoxy, 2'-NH
2 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'-N
3), 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.
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INTRODUCTION
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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.
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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).
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MATERIALS AND METHODS
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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 MgCl
2, 10 mM NaCl and 2 mM spermidine, with templates
at 10
–7 M and RNAPs at 2
x 10
–7 M. All NTPs were
used at 0.5 mM, and transcripts were labeled by inclusion of
1% (v/v) 800 Ci/mM [
-
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
x TBE gels, and visualized on a Molecular
Dynamics phosphorimager.
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RESULTS
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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).
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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.
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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).
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DISCUSSION
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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.
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ACKNOWLEDGEMENTS
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This work was supported by NIH grant GM52522 (to R. S.) and
funds from the Welch Foundation.
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REFERENCES
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