The EMBO Journal vol.14 no.18 pp.4609-4621, 1995 A mutant T7 RNA polymerase as a DNA polymerase Rui Sousa1 and Robert Padilla in dNTP Km (Ricchetti and Buc, 1993) and T7 DNA- directed RNA polymerase (RNAP) can also use RNA as Department of Biochemistry, University of Texas Health Science a template (Konarska and Sharp, 1989). These are not Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, exceptional observations, since it is a general property of TX 78212, USA polymerases that they display relaxed template specificity, 'Corresponding author at least in vitro. While template specificity may be relaxed, polymerase substrate specificity is normally extremely We have identified a T7 RNA polymerase (RNAP) stringent. T7 DNAP, for example, displays at least 2000- mutant that efficiently utilizes deoxyribonucleoside tri- fold selectivity for dNTPs over rNTPs, even in Mn2+ phosphates. In vitro this mutant will synthesize RNA, buffer, which relaxes the ability of the polymerase to DNA or 'transcripts' of mixed dNMP/rNMP composi- discriminate between dNTPs and ddNTPs (Tabor and tion depending on the mix of NTPs present in the Richardson, 1989). The structural determinants of such synthesis reaction. The mutation is conservative, stringent specificity remain undefined. changes Tyr639 within the active site to phenylalanine We report here the identification of mutant T7 RNAPs and does not affect promoter specificity or overall that display the ability to use dNTPs. The mutations occur activity. Non-conservative mutations of this tyrosine in Tyr639 within motif B (Delarue et al., 1990) of T7 also reduce discrimination between deoxyribo- and RNAP. Two observations impelled us to examine the ribonucleoside triphosphates, but these mutations also substrate discrimination and miscoding properties of these cause large activity reductions. Of 26 mutations of mutants. It had been found that mutations in the corres- other residues in and around the active site examined ponding conserved tyrosine in DNAP I increased mis- none showed marked effects on rNTP/dNTP dis- coding (Polesky et al., 1990; Carrol et al., 1991). It was crimination. Mutations of the corresponding tyrosine also found that transcripts synthesized by a T7 RNAP in DNA polymerase (DNAP) I increase miscoding, Y639F mutant in vivo yielded 33-50% of the protein per though effects on dNTP/rNTP discrimination for the transcript compared with transcripts synthesized by the DNAP I mutations have not been reported. This con- wild-type enzyme (Makarova et al., 1995). The latter served tyrosine may therefore play a similar role in phenotype was unique to the Y639F mutant amongst a many polymerases by sensing incorrect geometry in number of other active site mutants examined for in vivo the structure of the substrate/template/product due to expression and indicated that Y639F transcripts contained inappropriate substrate structure or mismatches. T7 a defect that led to their being inefficiently translated. RNAP can use RNA templates as well as DNA templates These observations implied that mutations in Tyr639 and is capable of both primer extension and de novo might cause increased misincorporation, either increased initiation. The Y639F mutant retains the ability to use mismatch synthesis (miscoding) or incorporation of sub- RNA or DNA templates. Thus this mutant can display strates of inappropriate structure. We have therefore de novo initiated or primed DNA-directed DNA characterized the ability of the Y639 mutants, as well as polymerase, reverse transcriptase, RNA-directed a large number of other active site mutants, to miscode RNA polymerase or DNA-directed RNA polymerase or to use dNTPs in both Mg2+ and Mn2+ buffers. Our activities depending simply on the templates and sub- results point to a specialized role for Tyr639 in T7 RNAP strates presented to it in the synthesis reaction. (and the corresponding tyrosine in other polymerases) in Keywords: mutagenesis/RNA polymerase/substrate speci- ensuring that substrates to be added to the growing nucleic ficity/transcription acid have the correct structure. They reveal that both transcript and substrate structure affect the efficiency with which the transcript is extended. They show that the Introduction restriction of unprimed initiation to RNAPs is not due to an intrinsic property of ribo- versus deoxyribonucleotides, One classification of nucleic acid polymerases relies on but simply to the selectivity of the polymerase active site. their different template specificities (RNA or DNA), They also present researchers with a novel reagent that substrate specificities (rNTPs or dNTPs) and mode of expands the structural range of nucleic acids that can be initiation (de novo or primed). These designations usually enzymatically synthesized in vitro. refer to the template and substrate specificities displayed in vivo during the fulfillment of a polymerase's biological Results function. In vitro, polymerases can display novel activities, albeit with reduced efficiency and/or under non-physio- Structure of the transcripts synthesized by Y639F logical conditions. Escherichia coli DNA-directed DNA and the wild-type enzyme with rNTPs and dNTPS polymerase (DNAP) I, for example, can use RNA as a Figure 1 shows transcription reactions carried out with template, though with a concomitant -100-fold increase the wild-type enzyme or the Y639F mutant polymerase K Oxford University Press 4609 R.Sousa and R.Padilla A BIC FIG HTI J IK N|O R|S T|U X geneous sequence abortive transcripts or 59 base run-off DIEI I I LIM PIQ VIW Pol: MWTMu WT Mu WT M.lu WT M. WT M.u WT M. WTM.lWTuWT Mu WT MIu WT Mu WTM.u products made and instead long poly(G) transcripts, as in rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP are NTPs: rATP dATP rATP rATP rATP rATP rATP rATP dATP dATP lane A, made. Adding dATP to reactions lacking rATP rCTP rCTP rCTP dCTP rCTP rCTP rCTP rCTP dCTP dCTP I rUTP rUTP rUTP rUTP rUTPI dTTP dTTP rUTP dTTP does not change the transcripts produced by the wild-type Rutlo - M enzyme (lane G). However, with the mutant enzyme we observe that addition of dATP (lane H) allows synthesis of a long run-off transcript, as well as synthesis of heterogeneous sequence abortive transcripts that do not co-migrate with the poly(G) transcripts. Observation of an abortive transcript in lane H running near the position of the 4H band in lane D confirms extension of the GGG trimer with an A, but note that the major 4mer transcript in lane H migrates close to, but not precisely with, the major 4mer in lane D or in adjacent lane I. This is consistent with the expectation that these 4mers will have identical sequence and length, but different structure (i.e. ._a rGrGrGrA in lanes D and I, rGrGrGdA in lane H). It should also be noted that some poly(G) transcript .7 ._w synthesis is observed in lane H. For example, in lane H we observe both a heterogeneous sequence 4mer migrating near the _=11111o ..:. 4H position and a smaller amount of 4mer band migrating ,LH 04 :-W 4 at the _w 4G position. When four rNTPs are present (lane C or D) synthesis of poly(G) transcripts is more completely suppressed. This indicates that dATP is utilized by Y639F, X - but not as efficiently as rATP. -,- Ck When rCTP is omitted from the reaction, transcripts .. terminate predominately at the 6mer length, because rCMP is normally first incorporated at position 7 (lanes I and J). Fig. 1. Structure of transcription products produced by Y639F and Addition of dCTP does not allow extension of the wild-type T7 RNAP in the presence of various combinations of rNTPs 6mer and dNTPs. The template was pT75 (Tabor and Richardson, 1985) cut in reactions with the wild-type enzyme (lane K). However, with HindIH so that transcription from its T7 promoter generated a 59 addition ofdCTP to reactions with Y639F allows extension base run-off transcript. Electrophoresis was on a 20% polyacrylamide- beyond the 6mer length and synthesis of the run-off 6 M urea gel. Plasmid and polymerases were at concentrations of transcript (lane L). Again, the following should be noted: l0e M and NTP concentrations were 0.5 mM (all rNTPs and dTTP), (i) transcripts larger than 6 bases do not 1 mM (dATP, dGTP) or 5 mM (dCTP). [,y-32P]GTP was added to co-migrate with radiolabel the transcription products. Wild-type (WT) or Y639F mutant their counterparts in lane C or D, consistent with the (Mu) polymerases and NTPs used are as indicated. Poly(rG) products expected structural difference despite length and sequence of various sizes are labeled in lane a (2G, 3G, etc.) and heterogeneous identity; (ii) there is more termination at the 6- and 7mer sequence abortive transcripts of different lengths are indicated by 4H, points in lane L than in lane C or D, that 5H, etc. in lane C. Lanes Q-T are a 10-fold longer exposure of lanes indicating Y639F M-P. uses dCTP well, but not as efficiently as it utilizes rCTP. In lanes M and N UTP was omitted from the reactions. Lanes Q-T show a 10-fold longer exposure of lanes M- and a T7 d10 promoter template. Transcription by T7 P. Within the set of four NTPs UTP is unique on this RNAP, like other RNAPs, is characterized by an initial, template, since it first becomes incorporated into the poorly processive 'abortive' phase of transcription during transcript at the 13 base position. This corresponds to a which the short, nascent transcript frequently dissociates transcript length subsequent to the transition from abortive from the ternary complex. When the transcript reaches a to processive transcription. As a consequence of this length of ;-9 bases transcription becomes highly processive transition the ternary complex becomes more stable and the transcript becomes stably associated with the (Martin et al., 1988).