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Nucleic Acids Research Pages 3550-3554  

Transcription of RNA templates by T7 RNA polymerase
Introduction
Materials And Methods
   Materials
   Transcription assays
Results
   Choice of the synthetic short templates
   T7 RNA polymerase is able to transcribe RNA from RNA+18 and RNA+1 templates
   The ability of T7 RNA polymerase to transcribe RNA templates is not sequence-dependent
Discussion
   T7 RNA polymerase is processive on an RNA template
   T7 RNA polymerase initiates on an RNA template
   T7 RNA polymerase is able to transcribe an RNA-RNA duplex
   The non-template strand may influence transcription efficiency
   RNA-dependent RNA polymerase properties of T7 RNA polymerase
Acknowledgements
References

Transcription of RNA templates by T7 RNA polymerase

Transcription of RNA templates by T7 RNA polymerase

Nadège Arnaud-Barbe, Valérie Cheynet-Sauvion, Guy Oriol, Bernard Mandrand, François Mallet*

Unité Mixte 103 CNRS-bioMérieux, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon, Cédex 07, France

Received March 31, 1998; Revised and Accepted June 23, 1998

ABSTRACT

Although highly specialized, T7 RNA polymerase seems to possess a large range of DNA- and RNA-dependent properties. To study such flexibility, we determined the ability of T7 RNA polymerase to transcribe chimeric DNA-RNA and RNA templates following initiation at a double stranded DNA promoter. We have found that T7 RNA polymerase is able to initiate on RNA templates, was processive, and was able to use templates containing RNA-RNA duplexes under standard transcription conditions. Implications of remnants of such RNA-dependent activities for T7 DNA-dependent polymerase are discussed.

INTRODUCTION

The bacteriophage T7 DNA-dependent RNA polymerase represents a useful model for studying transcription mechanisms. It is a monomeric enzyme which requires no auxiliary transcription factors for activity (1). The gene encoding T7 RNA polymerase has been cloned and the enzyme may be overexpressed in bacteria (2-4). Moreover, knowledge of its tertiary structure facilitates rational mutagenesis strategies (5).

T7 RNA polymerase is a highly specific enzyme (6): it recognizes specific promoters by contacts both on the template and the non-template strands. After binding to the promoter, repeated cycles of initiation and product release result in the accumulation of short abortive products. When the nascent RNA achieves a length of 8-12 nt, the T7 RNA polymerase undergoes a conformational change and enters the highly processive elongation phase. Termination occurs when the enzyme meets a terminator sequence or when it reaches the 5[prime] end of a linearized template.

Although T7 RNA polymerase functions in vivo to transcribe the bacteriophage T7 genome, it will also transcribe a variety of DNA templates that contain T7 promoters in vitro. Like the related T3 and SP6 RNA polymerases, it is very active on supercoiled and linear DNA templates and thus is routinely used to synthesize RNA for various biochemical applications (7) and, more recently, as components in multi-enzyme monothermal nucleic acid amplification systems like Nucleic Acid Sequence-Based Amplification (8), Self-Sustained Sequence Replication (9) and Transcription-Mediated Amplification (10). Furthermore, Milligan et al. (11) showed that T7 RNA polymerase does not require duplex DNA for activity, but can produce large amounts of RNA from synthetic short DNA templates that contain a double stranded promoter region and a single stranded region downstream. The same properties were observed for SP6 RNA polymerase (12), although the relative efficiencies of transcription on such templates is not clear (11-13). It has also been reported that T7 RNA polymerase has promoter-independent properties on DNA templates as it is able to extend RNA primers (14).

In addition to the DNA-dependent activities noted above, T7 RNA polymerase also has some RNA-dependent properties, as it is able to replicate certain RNA hairpin loops that are not replicated by other RNA polymerases (15-17), and is able to elongate self-complementary RNA templates (18). Although T3 RNA polymerase has been shown to transcribe a chimeric DNA/RNA template having a duplex DNA T3 promoter joined to a duplex RNA template, with the transition between DNA and RNA six bases downstream from the promoter sequence (19), no such activity has yet been described for T7 RNA polymerase.

The highly specialized T7 RNA polymerase thus seems to possess a large range of DNA- and RNA-dependent properties. To study such flexibility, we determined the ability of T7 RNA polymerase to transcribe chimeric DNA-RNA and RNA templates following initiation at a double stranded DNA promoter. We have determined that T7 RNA polymerase is processive on a variety of chimeric templates in which the transition between DNA and RNA occurs 18 bases downstream from the promoter region. Moreover, it initiates on templates having different RNA sequences in the initiation region. Finally, it is able to melt an RNA-RNA duplex during the initiation and elongation phases.

MATERIALS AND METHODS

Materials

T7 RNA polymerase (specific activity of 200 U/µg) was purified by immobilized metal ion affinity chromatography (4).

The oligonucleotides were purchased from Eurogentec or synthesized with a 394 Applied Biosystem DNA/RNA synthesizer using cyanoethyl-phosphoramidite chemistry on a 1 µmol scale according to the manufacturer's protocol; all the reagents and standards were from Applied Biosystem (Foster City, USA). DNA oligonucleotides were purified either by reverse phase on HPLC or on a Polypaq Cartridge. RNA oligonucleotides were purified by electrophoresis in polyacrylamide gels. The oligo-nucleotides used as templates are described in Figure 1. Probes P13 (3[prime]-ATTGCGAGGTTCATGT-5[prime]) and P2 (3[prime]-GTGAGTACCAATACCG-5[prime]) recognized the 3[prime] end of the groups 1 and 3 or of the group 2 transcripts (see below), respectively; they were labeled with [[gamma]-32P]ATP by T4 polynucleotide kinase or with [[alpha]-32P]ddATP by terminal deoxynucleotide transferase.


Figure 1. Oligonucleotides used as templates in the transcription assays. (A) The top strand is the non-template strand and the bottom strand is the template strand. Thick lines represent the consensus double stranded DNA promoter region (25); fine lines are DNA regions; dashed lines are RNA regions; +1 corresponds to the start site of transcription; +18 corresponds to the 18 base downstream from the start site; ss DNA, single stranded DNA template; dsho DNA, double stranded homoduplex DNA template; ss RNA+18, single stranded RNA+18 template; dshe RNA+18, double stranded heteroduplex RNA+18 template; dsho RNA+18, double stranded homoduplex RNA+18 template; ss RNA+1, single stranded RNA+1 template; dshe RNA+1, double stranded heteroduplex RNA+1 template; dsho RNA+1, double stranded homoduplex RNA+1 template. (B) The sequence of the template strand is indicated. The promoter sequence is underlined; the +1 to +6 region is in bold letters. Templates from group 1 consist of a double stranded DNA promoter sequence followed by a +1 to +6 consensus initiation region (25) and a 26 base downstream sequence; templates from group 2 consist of a double stranded DNA promoter sequence followed by a non-consensus initiation region and a 27 base downstream sequence; templates from group 3 consist of the same initiation sequence as group 2 templates followed by the same downstream sequence as group 1 templates. The downstream sequence of group 1 and group 3 templates have been randomly chosen and the absence of secondary structure was checked using a commercial program (OLIGO 4.03, National Bioscience, Plymouth, MN). The downstream sequence of group 2 templates was selected as part of a gene ([beta]-lactamase).

Transcription assays

Reactions were carried out in a volume of 20 µl containing 40 mM Tris-HCl, 1 mM spermidine, 50 µg/ml bovine serum albumin, 0.01% (v/v) Triton X-100, 80 mg/ml PEG 8000, 1 µl RNAguard (Pharmacia), 6 mM magnesium acetate, 1011 copies (0.17 pmol) template and non-template strands, NTPs and labeled NTPs as indicated. When the labeled NTP was [[alpha]-32P]ATP, 0.4 mM UTP, CTP and GTP, 12.5 µM ATP and 0.5 µCi [[alpha]-32P]ATP (New England Nuclear-Dupont, 800 Ci/mmol) were used; when the labeled NTP was [[alpha]-32P]UTP, 0.4 mM ATP, CTP and GTP, 12.5 µM ATP and 0.5 µCi [[alpha]-32P]UTP (Amersham, 400 Ci/mmol) were used; when the labeled NTP was [[gamma]-32P]GTP, 0.4 mM ATP, UTP, CTP and GTP, and 20 µCi [[gamma]-32P]GTP (Amersham, 5000 Ci/ mmol) were used.

The samples were heated for 5 min at 70°C and slowly cooled to 37°C to allow annealing of template and non-template strands. Then, 260 ng T7 RNA polymerase were added and the samples were incubated for 1 h at 37°C. Reactions were stopped by adding an equal volume of 2× loading buffer (90% formamide, 25 mM EDTA, 0.02% xylene cyanol, 0.02% bromophenol blue), followed by heating at 95°C for 5 min. The products were resolved by electrophoresis in denaturing 20% polyacrylamide gels and visualized by autoradiography.

For northern blot analysis, the reactions were performed under the same conditions without labeled nucleotides and the samples were transferred to a nylon membrane (Appligene); products were detected by hybridization to the relevant labeled probe and visualized by autoradiography.

[[gamma]-32P]GTP labeled transcription products and appropriate dilutions of a stock of [[gamma]-32P]GTP were run on denaturing 23% polyacrylamide gels and scanned on a Storm 860 PhosphorImager (Molecular Dynamics) for quantitation. The values determined for the standard dilutions were used to plot a curve allowing conversion of PhosphorImager units into moles of transcripts.

RESULTS

Choice of the synthetic short templates

Three kinds of short synthetic templates, each containing a double stranded T7 DNA promoter, were designed to determine whether T7 RNA polymerase is able to use an RNA template for promoter-dependent transcription (Fig. 1A). The first type of template (RNA+18) was designed to study the ability of T7 RNA polymerase to transcribe an RNA template during the processive phase. These templates consist of a double stranded DNA promoter followed by an RNA region in which the transition from DNA to RNA occurs 18 bases downstream from the promoter sequence. This design was based on the observation that T7 RNA polymerase undergoes the transition from an unstable initiation complex to a stable elongation complex after the synthesis of 8-12 nt of nascent RNA. The second type of template (RNA+1) was designed to study the ability of T7 RNA polymerase to utilize an RNA template strand during the initiation phase. It consisted of a double stranded DNA promoter joined to an RNA template downstream from the start site at +1. The third type of template (DNA) consisted of a double stranded DNA promoter followed by a DNA transcribed region (the natural situation).

To study the influence of the non-template strand, DNA, RNA+18 and RNA+1 templates were single stranded (ss), double stranded heteroduplex (dshe) or double stranded homoduplex (dsho) in the transcribed region.

To determine the influence of base composition on transcription efficiency, different sequences were employed, resulting in three groups (Fig. 1B). These templates contained either a consensus (group 1) or a variant initiation region from +1 to +6 (groups 2 and 3) and two different downstream sequences (groups 1 and 3 versus group 2).

T7 RNA polymerase is able to transcribe RNA from RNA+18 and RNA+1 templates

The ability of T7 RNA polymerase to transcribe the templates described above was evaluated using 260 ng of enzyme per reaction, corresponding to a non-saturating concentration of enzyme as the transcription reaction was shown linear within the 200-700 ng enzyme range on DNA template (data not shown). Qualitative analysis of transcription from the templates described above was determined by [[alpha]-32P]ATP incorporation and by northern blot analysis. Runoff, abortive and intermediate products were observed with all templates (Fig. 2). They were all due to transcription from the T7 promoter since they did not appear in absence of a functional promoter (Fig. 2). The specific runoff RNAs (Fig. 3A) were 32-34 nt in length, consistent with the known property of T7 RNA polymerase to add one or two nucleotides at the 3[prime] end of the transcript (20). Abortive transcripts had a maximum length of 12 nt. Synthesis of intermediate products due to premature termination of transcription were observed all along the template, and especially when approaching the 5[prime] end of the template. Additional 50-60 nt products were observed to be synthesized from the RNA+18 and RNA+1 templates. These products may correspond to hybrids formed between the template strand and the transcript since (i) they were due to specific transcription, (ii) they were not detected by northern blot and (iii) they appeared in proportion to runoff transcripts. The absence of such products following transcription of DNA templates is consistent with the lower stability of DNA-RNA hybrids as opposed to RNA-RNA hybrids.


Figure 2. Autoradiogram of transcription products synthesized by T7 RNA polymerase from group 1 templates. The incorporation of [[alpha]-32P]ATP was performed as described in Materials and Methods. The assays (templates) consisted in transcription of single stranded (ss), double stranded heteroduplex (dshe) and double stranded homoduplex (dsho) DNA, RNA+18 and RNA+1 templates. The negative controls corresponded to transcription of each strand of the templates without the complementary strand. P, non-template strand of -17 to +1 promoter region; t, DNA, RNA+18 or RNA+1 template strand; nt, DNA, RNA+18 or RNA+1 non-template strand; M, molecular weight marker (nucleotides).


Figure 3. Northern blot analysis of transcription by T7 RNA polymerase from (A) group 1, (B) group 2 and (C) group 3 templates. Northern blot was performed as described in Materials and Methods with the radiolabeled probe P13 for groups 1 and 3 transcripts and P2 for group 2 transcripts. Specific runoff transcripts are indicated by an arrow; note that only one single band was observed due to the gel resolution. The higher band corresponds to the non-template strand when present.

Quantitative analysis of transcription from DNA, RNA+18 and RNA+1 templates by [[gamma]-32P]GTP incorporation allowed comparison of the transcription process on these different templates (Table 1). The most efficient transcription led to synthesis of a 250-fold higher amount of runoff RNA than the less efficient transcription, corresponding to 800 and 3 pmol of runoff RNA from 10 pmol of ss RNA+18 and dshe RNA+1 templates, respectively. Surprisingly, the amount of runoff products was 2-3-fold higher on RNA+18 than on DNA templates. About 170-800 pmol of runoff RNA were synthesized from 10 pmol of RNA+18 templates, as compared to 80-240 from DNA templates. Moreover, the amount of abortive and intermediate transcripts increased to about the same extent, corresponding to a 2-3-fold increase in the number of initiation events. Hence, transcription efficiency (runoff products/total products) was similar on DNA and RNA+18 templates (1/8-1/16). Thus, T7 RNA polymerase was processive on an RNA template if initiation occurred on DNA. The number of initiation events decreased only 2-fold on RNA+1 templates as compared to DNA templates. However, the relative percentage of abortive products increased, reducing the transcription efficiency (1/108-1/227). About 3-13 pmol of runoff RNA were synthesized from 10 pmol of RNA+1 templates. We therefore conclude that T7 RNA polymerase is able to initiate at a promoter in which the template consists of RNA downstream from +1.

The non-template strand influenced the efficiency of transcription from DNA, RNA+18 and RNA+1 templates. Indeed, transcription from single stranded templates provided a 1.4-2.3-fold increase in the number of initiation events and a 2-4.5-fold increase in the amount of runoff RNA.

The ability of T7 RNA polymerase to transcribe RNA templates is not sequence-dependent

To determine whether the ability of T7 RNA polymerase to use an RNA template depends upon the transcribed sequence, we utilized templates containing different sequences in the entire transcribed region (group 2) or only in the initiation region (+1 to +6) (group 3).

Table 1. Quantitation of transcription products synthesized by T7 RNA polymerase from group 1 templates
Templates Runoff
(pmol)		 Abortive
(pmol)		 Intermediate
(pmol)		 Initiation
eventsa		 Efficiencyb
ss DNA 240 1760 416 242 1/10
dsho DNA 80 1312 128 152 1/18
ss RNA+18 800 4480 1216 650 1/8
dshe RNA+18 208 2496 384 309 1/14
dsho RNA+18 176 2240 304 272 1/16
ss RNA+1 13 1280 112 141 1/108
dshe RNA+1 3.2 576 32 61 1/227
dsho RNA+1 6.4 1024 64 110 1/157
Reactions were performed as described in Materials and Methods. The results indicated are for transcription from 10 pmol of template. Quantitation of runoff, abortive and intermediate transcripts have a precision of ±20% as determined by quantitation of replicate assays.
aThe number of initiation events (n) indicated that 2.7 pmol (260 ng) of T7 RNA polymerase initiate transcription n times from a single copy of template.
bTranscription efficiency corresponds to the number of initiation events that leads to runoff RNA versus the total number of initiation events performed during the reaction from one copy of template.

Transcription patterns from the group 2 and 3 templates were similar to those from group 1 templates, although some differences existed. Runoff RNAs were synthesized from DNA and RNA+18, as visualized by [[alpha]-32P]UTP incorporation (data not shown) and by northern blot (Fig. 3B and C), but runoff RNAs from RNA+1 templates were detected only by northern blot analysis, with the exception of the single stranded RNA+1 template from group 2. This may be due to the limited sensitivity of RNA detection using [[alpha]-32P]UTP incorporation, since in our conditions, northern blot analysis is 40 times more sensitive than [[alpha]-32P]UTP incorporation. The pattern of abortive products, characterized by the large production of 3-6 nt RNAs, was quite different from that obtained with the group 1 templates. Finally, intermediate and additional 50 nt products were still observed.

Quantitation of RNA synthesized from DNA and RNA+18 templates could be obtained by [[gamma]-32P]GTP incorporation but quantitation from RNA+1 templates was not possible (Table 2). The transcription efficiency from group 2 and group 3 templates was always lower than from group 1 templates, and although the amount of runoff RNA was of the same order for each DNA and RNA+18 templates, the efficiency of transcription varied significantly and was quite low. We can notice that ~95% of abortive products were 3-6 nt RNAs as opposed to a lesser ratio from group 1 templates (data not shown). No dramatic difference in transcription efficiency between single stranded and double stranded group 2 templates was observed.

DISCUSSION

T7 RNA polymerase is processive on an RNA template

As previously observed with T3 RNA polymerase (19), T7 RNA polymerase is found to be processive on an RNA template following initiation at a double stranded promoter. The transition from DNA to RNA in the template strand at +18 was efficiently bypassed by the T7 RNA polymerase elongation complex, as no premature termination around +18 was detected. The presence of an RNA template in the elongation region did not appear to affect initiation, the conformational change or the elongation steps. In fact, an increased number of initiation events with group 1 RNA+18 templates might reflect an increased availability of T7 RNA polymerase after one round of transcription suggesting that release of the ternary elongation complex at the end of the template may be favoured by the sequence and/or the structure of the RNA region. The relative percentage of abortive products, and especially of 3-6 nt RNAs, increased with group 2 and group 3 templates. This may be due to the initiation sequence in these templates, which is predicted to lead to the formation of weak dA.rU bonds between the newly synthesized RNA and the template strand, thus favouring the release of the initiation complex (21). As expected, no qualitative difference in the initiation process was detected between DNA and RNA+18 templates for each template group since initiation occurred within the same DNA sequence context.

Table 2. Comparison of transcription by T7 RNA polymerase from different templates
Templates Group (1) Group (2) Group (3)
Runoff (pmol) Efficiencya Runoff (pmol) Efficiencya Runoff (pmol) Efficiencya
ss DNA 240 1/10 192 1/31 149 1/43
dsho DNA 80 1/18 102 1/19 67 1/133
ss RNA+18 800 1/8 326 1/45 253 1/49
dshe RNA+18 208 1/14 99 1/65 61 1/106
dsho RNA+18 176 1/16 358 1/31 ND ND
ss RNA+1 13 1/108 NA NA NA NA
dshe RNA+1 3.2 1/227 NA NA NA NA
dsho RNA+1 6.4 1/157 NA NA ND ND
Reactions were performed as described in Materials and Methods. The results indicated are for transcription from 10 pmol of templates. Quantitation of runoff, abortive and intermediate transcripts have a precision of ± 20% as determined by quantitation of replicate assays. NA, not applicable; ND, not determined.
aTranscription efficiency corresponds to the number of initiation events that leads to runoff RNA versus the total number of initiation eventsperformed during the reaction from one copy of template.

T7 RNA polymerase initiates on an RNA template

T7 RNA polymerase was able to initiate and transcribe promoters having RNA downstream from +1. Initiation on an RNA template did not prevent the transition to the elongation phase even though a highly stable duplex between the template strand and the newly synthesized RNA is expected to form on these templates. However, either processivity decreased, or more likely, abortive transcription increased on these templates, perhaps due to a less efficient isomerization. The production of specific transcripts from a variety of such promoters points out that transcription of the RNA template is not sequence-dependent, but that the overall efficiency may depend on base composition, as is observed with DNA templates.

T7 RNA polymerase is able to transcribe an RNA-RNA duplex

T7 RNA polymerase was able to melt an RNA-RNA duplex during both the elongation phase and the initiation phase. Thus, processivity was not altered on the dsho RNA+18 template even though a stable RNA-RNA duplex must be bypassed by the ternary elongation complex on this template. Furthermore, no increased abortive transcription was observed with the dsho RNA+1 template in which a stable RNA-RNA duplex in the initiation region must be melted open. Transcription of dshe and dsho templates was in general similar. However, the presence of an RNA-RNA duplex in group 2 RNA+18 templates appeared to favour stability of the ternary complex since more runoff products were synthesized on dsho than on dshe RNA+18 templates.

The non-template strand may influence transcription efficiency

The presence of a non-template strand in the transcribed region influenced transcription of DNA, RNA+18 and RNA+1 templates by T7 RNA polymerase. Indeed, transcription yield increased on single stranded templates downstream from +1, characterized by synthesis of higher amount of runoff RNA and by the fact that initiation events led more often to runoff products. Thus, transcription appears to be improved on DNA templates when the transcribed region is single stranded. This is in agreement with Diaz et al. (13), who showed that T7 RNA polymerase formed a highly stable complex on single stranded DNA templates. However, we cannot exclude that the differences between double stranded and single stranded templates may reflect release of transcripts at the end of the templates. The preference for single stranded template is not a general rule since it was not observed with the group 2 templates. Thus, the entire template sequence may influence the relative use of double stranded versus single stranded templates. This would explain conflicting results showing either a better transcription yield on full-length double stranded template (12), or no different transcription yield with both kinds of templates (11).

RNA-dependent RNA polymerase properties of T7 RNA polymerase

We have found that T7 RNA polymerase is able to initiate on RNA templates, is processive and is able to use templates containing RNA-RNA duplexes under standard transcription conditions. The transcription yield was only 10-100-fold lower than on DNA templates, in these experimental conditions. Replication of RNA hairpins (15-17) and elongation of self-complementary RNA (18) by T7 RNA polymerase have previously been described. The ability of T7 RNA polymerase to catalyse RNA replication and RNA-directed transcription may open new fields of application, in particular in nucleic acid amplification technologies. Moreover, remnants of such RNA-dependent activities for DNA-dependent polymerases such as T7 RNA polymerase and Escherichia coli DNA polymerase I (22) supports the hypothesis of divergent evolution for these nucleotide polymerases.

The common right-handed shape shared by the tertiary structure of crystallized polymerases (23), including T7 RNA polymerase and a recently described RNA-dependent RNA polymerase (24), may allow a mutagenesis strategy to study the RNA-dependent activity of T7 RNA polymerase.

ACKNOWLEDGEMENTS

We are grateful to Christelle Tora and Viviane Monnot for oligonucleotide synthesis, and Françoise Guillou-Bonnici for providing us with some templates. We thank Karina Dutter for excellent technical assistance. N.A.-B. was supported by a doctoral fellowship from the Fondation Marcel Mérieux.

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*To whom correspondence should be addressed. Tel: +33 472 728 358; Fax: +33 472 728 533; Email: fmallet{at}ens-bma.cnrs.fr

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