T7 DNA Template Research Articles

Maximizing the mRNA productivity for in vitro transcription by optimization of fed-batch strategy

In vitro transcription (IVT) is the main manufacturing method to produce mRNA vaccines. In this study, a fed-batch strategy was systematically optimized for IVT process with dual goals of achieving a high reaction rate and maximizing the final mRNA yield simultaneously. Initially various experimental conditions were investigated including Mg2+, nucleotide triphosphates (NTP), dithiothreitol (DTT), spermidine, as well as the temperature and ionic strength. It was found that the concentrations of Mg2+ and NTP had a significant impact on IVT process. Subsequently, under the optimized conditions, dividing the IVT reaction into three distinct phases was proposed to enable more efficient transcription. By optimizing the concentrations of Mg2+ and NTP in two replenishment processes, our fed-batch strategy resulted in the production of 367.8 μg of mRNA with reduced dsRNA byproducts within 180 min. This was achieved under conditions of a final volume of 30 μL, 250 U T7 RNA polymerase (RNAP), and 2 μg DNA template. In conclusion, the IVT process using a fed-batch approach, rather than an excessive one-time NTP input, is an efficient method to improve productivity given a fixed amount of T7 RNAP and DNA template.

Isothermal detection of RNA transcription levels using graphene oxide-based SYBR Green I fluorescence platform

Abstract An isothermal graphene oxide (GO)-based SYBR Green I fluorescence platform for sensitive detection of RNA transcription levels is proposed. Briefly, a synthesized T7 DNA template was transcribed by T7 RNA polymerase, to produce many copies of single-stranded (ss) RNA transcripts. The ssRNA transcripts were then hybridized with a label-free ssDNA probe which alone will be adsorbed on the GO surface. The resultant double-stranded (ds) RNA:DNA hybrids were incubated with GO and SYBR Green I. Then the hybrid binding with SYBR Green I emits enhanced fluorescence for detection. Experimental results show that the detection limit of the method is 0.5 pmol/L of T7 DNA template. A calibration curve with a linearity range of 0.5 pmol/L to 5 nmol/L is established, thus, making quantitative analysis feasible. The method may become a powerful tool for RNA transcription detection due to its sensitivity, rapidity and convenience.

The Transition to an Elongation Complex by T7 RNA Polymerase Is a Multistep Process

During the transition from an initiation complex to an elongation complex (EC), T7 RNA polymerase undergoes major conformational changes that involve reorientation of a "core" subdomain as a rigid body and extensive refolding of other elements in the 266 residue N-terminal domain. The pathway and timing of these events is poorly understood. To examine this, we introduced proline residues into regions of the N-terminal domain that become alpha-helical during the reorganization and changed the charge of a key residue that interacts with the RNA:DNA hybrid 5 bp upstream of the active site in the EC but not in the initiation complex. These alterations resulted in a diminished ability to make products >5-7 nt and/or a slow transition through this point. The results indicate that the transition to an EC is a multistep process and that the movement of the core subdomain and reorganization of certain elements in the N-terminal domain commence prior to promoter release (at 8-9 nt).

Incomplete Factorial and Response Surface Methods in Experimental Design: Yield Optimization of tRNATrp from In vitro T7 RNA Polymerase Transcription

We have studied the yield of Escherichia coli tRNA(Trp) obtained from in vitro T7 RNA polymerase transcription using incomplete factorial and response surface methods. Incomplete factorial experiments were first used to estimate the relative impact of six variables on the yield of tRNA(Trp). Fifteen trials were performed according to a balanced and randomized design. The correlation between observed yield and all experimental variables was identified by stepwise multiple linear regression analysis. The concentrations of T7 RNA polymerase, DNA template, NTP and MgCl2 proved to be significantly correlated with the yield of tRNA(Trp). We then optimized the yield with respect to each of these four variables simultaneously with a designed, response surface experiment based on the Hardin-Sloane minimum prediction variance algorithm. Twenty experiments were performed, in duplicate, to sample the quadratic surface relating the yield to the four significant variables. Coefficients of the quadratic function with all two-factor interactions were evaluated by stepwise regression using least squares, and significant coefficients were retained. Partial differentiation of the resulting quadratic model showed it to possess an optimum. Transcription performed at the corresponding conditions yielded 6-fold more tRNA(Trp) than the initial conditions, confirming the predictive value of the experimentally determined response surface.

Processing of transfer RNA precursors in a wheat mitochondrial extract.

To investigate mechanisms for processing of plant mitochondrial RNAs, we studied the fate of wheat mitochondrial tRNA precursors in a homologous soluble extract. Artificial precursor transcripts were synthesized in vitro using T3 or T7 RNA polymerase and DNA templates containing wheat mitochondrial tRNA genes and flanking sequences. We found that the mitochondrial extract supports processing of precursors containing both native and chloroplast-like (Joyce, P. B. M., and Gray, M. W. (1989) Nucleic Acids Res. 77, 5461-5476) wheat mitochondrial tRNA sequences. Incubation of precursor transcripts with the extract results in processing of tRNAs via precise 5'- and 3'-endonucleolytic cleavages. However, these cleavages are not ordered in vitro because intermediates composed of 5'-leader + tRNA and tRNA + 3'-trailer are present simultaneously throughout the course of the reaction. Sequence analysis of processed products confirmed that endonucleolytic cleavages occur at the expected positions, generating tRNAs with 5'-phosphoryl and 3'-hydroxyl termini. The mitochondrial extract also contains a tRNA nucleotidyltransferase activity that adds -CCAOH termini to the 3'-ends of processed tRNAs. This cell-free RNA processing system provides the basis for biochemical characterization of the various enzymes involved in the production and maturation of plant mitochondrial tRNAs.

RNA chain elongation by Escherichia coli RNA polymerase : Factors affecting the stability of elongating ternary complexes

We have devised a method to follow the stability of individual ternary transcription complexes containing Escherichia coli RNA polymerase halted at many different sites along a DNA template during the transcription process. Studies of complexes formed with phage T7 DNA templates reveal at least three general classes of ternary complexes that differ dramatically in their properties. Complexes of one sort (normal complexes) are highly stable to dissociation and denaturation under a variety of solution conditions. They remain intact and active for up to 24 hours even in salt concentrations up to 1 M-K+. This suggests that they are stabilized to a significant extent by non-ionic interactions between RNA polymerase and the nucleic acids. We consider these to be the normal complexes formed during RNA chain elongation. Complexes of a second sort (release complexes) dissociate rapidly, releasing free RNA transcripts and active RNA polymerase. The rate of dissociation is substantially enhanced by elevated concentrations of K+, hence the interaction between RNA polymerase and nucleic acids in these complexes is stabilized predominantly by ionic interactions. However, release complexes are stabilized by millimolar concentrations of Mg2+, which as been implicated in stabilization of the binding of RNA to free RNA polymerase. These complexes are formed at DNA sequences that we refer to as release sites. Analysis of DNA sequences at release sites reveals that all share a common feature, the potential to form an RNA hairpin in the region just upstream from the actual 3' end of the released RNA. Experiments incorporating IMP in the transcript and blocking potential hairpin formation with DNA oligomers support a direct role for an RNA hairpin in triggering the release reaction. Changes in the 3'-proximal DNA sequences generally have little effect on the presence or rate of the release reaction, although there are significant exceptions. The results suggest that the presence of certain RNA hairpins in the region six to ten nucleotides upstream from the transcript growing point can trigger a substantial structural transition in the ternary transcription complex, forming a "release mode" complex from which transcript dissociation is facilitated. This release, mode complex may be a central intermediate in RNA chain termination. A final class of complexes (dead-end complexes) appear to be elongating complexes that have entered a state or conformation that is stable, but is blocked in resuming the normal elongation reaction. Such complexes bear active RNA polymerase, and can be restarted after limited pyrophosphorolysis. The structural elements that determine the formation of dead-end complexes are not yet known.

Association of RNA polymerase having increased Km for ATP and UTP with hyperexpression of the pyrB and pyrE genes of Salmonella typhimurium.

We investigated the transcription kinetics of RNA polymerase from an rpoBC mutant of Salmonella typhimurium which showed highly elevated, constitutive expression of the pyrB and pyrE genes as well as an increased cellular pool of UTP. When bacterial cultures containing an F' lac+ episome were induced for lac operon expression, the first active molecules of beta-galactosidase were formed with a delay of 73 +/- 3 s in rpo+ cells. The corresponding time was 104 to 125 s for cells carrying the rpoBC allele, indicating that this mutation causes a reduced RNA chain growth rate. In vitro the purified mutant RNA polymerase elongated transcripts of both T7 DNA and synthetic templates more slowly than the parental enzyme at a given concentration of nucleoside triphosphates. This defect was found to result from four- to sixfold-higher Km values for the saturation of the elongation site by ATP and UTP. The saturation kinetics of the RNA chain initiation step also seemed to be affected. The maximal elongation rate and Km for GTP and CTP were less influenced by the rpoBC mutation. Open complex formation at the promoters of T7 DNA and termination of the 7,100-nucleotide transcript showed no significant difference between the parental and mutant enzymes. Together with the phenotype of the rpoBC mutant, these results indicate that expression of pyrB and pyrE is regulated by the mRNA chain growth rate, which is controlled by the cellular UTP pool. The rate of gene expression is high when the saturation of RNA polymerase with UTP is low and vice versa.

Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase.

The function of lysyl residues of the sigma subunit of the RNA polymerase from Escherichia coli was investigated by chemical modification with trinitrobenzenesulfonic acid (TNBS). Following reaction with TNBS, analysis of the modified sigma indicated that trinitrophenylation was limited to the epsilon-amino groups of lysyl residues. Progressive loss in the activity of sigma followed increasing trinitrophenylation as assayed by the ability to stimulate RNA polymerase core enzyme in a reaction directed by T7 DNA. Modification of five lysyl groups resulted in the complete loss of sigma activity. Kinetic analysis indicated that one lysyl group is critical for the function of sigma. TNP-sigma was able to form a holoenzyme complex with a binding affinity comparable to that of sigma. Promoter recognition studies were done by using HindIII fragments from T5 DNA. The TNP-sigma core complex was unable to form a tight binary complex with the T5 promoters. Studies on RNA chain initiation were carried out by using d(A-T)n and T7 DNA templates. TNP-sigma was unable to stimulate RNA chain initiation by core polymerase. Limited proteolytic digests of TNP-sigma or sigma using Staphylococcus aureus V8 protease were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggested a change in the conformation of sigma following trinitrophenylation.

Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.

Preparations of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B. subtilis RNA polymerase holoenzyme II) that shows a unique promoter specificity with T7 bacteriophage DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I) and lacks the normal sigma subunit [Jaehning, J. A., Wiggs, J. L. & Chamberlin, M. J. (1979) Proc. Natl. Acad. Sci. USA 76, 5470-5474]. By heparin-agarose chromatography we have obtained holoenzyme II fractions that have no detectable holoenzyme I activity as judged by their failure to utilize promoter sites for holoenzyme I on any template we have tested. These fractions are far more active with B. subtilis DNA than with T7 DNA or other heterologous templates. This high degree of specificity has allowed identification of plasmids containing cloned fragments of B. subtilis DNA that bear strong promoter sites for holoenzyme II. These promoter sites are not used at all by B. subtilis RNA polymerase holoenzyme I. The specificity of holoenzyme II is dictated by a peptide of Mr 28,000 as judged by copurification of the peptide with specific holoenzyme II activity and by reconstitution of the holoenzyme II promoter specificity when the isolated peptide is added to B. subtilis core polymerase. Hence the 28,000 Mr peptide appears to be a sigma factor that determines a promoter specificity distinct from that of RNA polymerase holoenzyme I and all other known bacterial RNA polymerases.

Bacteriophage T7 DNA replication in vitro. Stimulation of DNA synthesis by T7 RNA polymerase.

Four T7 products, DNA polymerase, gene 4 protein, RNA polymerase, and DNA binding protein, have been purified from phage-infected cells. It has been previously shown (Hinkle, D. C., and Richardson, C. C. (1975) J. Biol. Chem. 250, 5523-5529; Kolodner, R., and Richardson, C. C. (1978) J. Biol. Chem. 253, 574-584) that two T7 products, DNA polymerase and gene 4 protein, catalyze extensive synthesis on duplex T7 DNA containing single strand breaks. However, the T7 DNA polymerase purified by our procedure does not efficiently contribute in this reaction, although the preliminary evidence suggests that this enzyme may be the native form of the DNA polymerase. Such inefficient T7 DNA synthesis is greatly augmented by adding the third T7 product, namely T7 RNA polymerase. This DNA synthesis apparently requires transcription, since each of the four rNTPs must be present. The rate of synthesis is increased about 2-fold by the addition of T7 DNA binding protein. In contrast to the results obtained when DNA synthesis is initiated at single strand breaks in a duplex DNA molecule, essentially none of the DNA synthesized in the presence of T7 RNA polymerase is covalently attached to the T7 DNA template. We postulate that in this in vitro system, T7 DNA replication is initiated using an RNA primer synthesized by the T7 RNA polymerase.

The effect of low substrate concentrations on the extent of productive RNA chain initiation from T7 promoters A1 and A2 by Escherichia coli RNA polymerase.

The extent of productive RNA chain initiation in vitro by Escherichia coli RNA polymerase holoenzyme from the bacteriophage T7 A1 and A2 promoters on purified T7 DNA templates has been investigated at very low concentrations of the ribonucleoside triphosphate substrates. As the concentration of ribonucleoside triphosphates in the reaction is decreased from 10 to 1 micro M, the extent of productive RNA chain initiation at these promoter sites drops precipitously at about 3 micro M. At 1 micro M substrate concentration, productive chain initiation from the A1 promoter does not occur even after extended incubation although the dinucleoside tetraphosphate pppApU is produced at a significant rate under these conditions. The reason for the inability of RNA polymerase to carry out productive RNA chain initiation at 1 micro M substrate concentration is not yet understood. The phenomenon is not due to substrate consumption, enzyme inactivation, or a requirement for a nucleoside triphosphatase activity in the reaction. The possibility is raised that there are additional nucleoside triphosphate binding sites on E. coli RNA polymerase which play some role in the process of productive RNA chain initiation.

Gene 4 protein of bacteriophage T7. Characterization of the product synthesized by the T7 DNA polymerase and gene 4 protein in the absence of ribonucleoside 5'-triphosphates.

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze extensive DNA synthesis on duplex phage T7 or PM2 DNA templates containing single strand breaks. A variety of physicochemical techniques have been used to characterize the DNA product synthesized in this reaction in the absence of ribonucleoside 5'-triphosphates. Pyknographic and sedimentation analyses reveal that all of the newly synthesized DNA is covalently attached to the template DNA. Analysis by electron microscopy shows the major portion of the product molecules synthesized on duplex T7 DNA templates to consist of a double-stranded branch attached to an intact template molecule. Using PM2 DNA templates, the predominant product consists of a double-stranded branch attached to the circular PM2 DNA template. Analyses of these product molecules indicate that DNA synthesis by the gene 4 protein and T7 DNA polymerase is initiated at single strand breaks in the duplex DNA and that synthesis is accompanied by extensive displacement of one of the parental strands. At later times in the reaction, a portion of the 3'-hydroxyl terminus of the newly synthesized DNA is displaced from the template by branch migration and is used as a primer by the DNA polymerase to copy the displaced 5' single-stranded parental strand to form a duplex branch.

Gene 4 protein of bacteriophage T7. Purification physical properties, and stimulation of T7 DNA polymerase during the elongation of polynucleotide chains.

With the use of an in vitro complementation assay to measure activity, the gene 4 protein of bacteriophage T7 has been purified 1000-fold to yield a nearly homogeneous protein. The purified gene 4 protein is a single polypeptide having a molecular weight of 58,000. In addition to being essential for T7 DNA replication in vivo and in vitro, the gene 4 protein is required for DNA synthesis by the purified T7 DNA polymerase on duplex T7 DNA templates. In the absence of ribonucleoside 5'-triphosphates, DNA synthesis by the gene 4 protein and the T7 DNA polymerase is dependent on phosphodiester bond interruptions containing 3'-hydroxyl groups (nicks) in the duplex DNA. The reaction is specific for the T7 DNA polymerase, but any duplex DNA containing nicks can serve as template. The Km for nicks in the reaction is 3 x 10(-10) M.

Thermodynamic limits on the size and size distribution of nucleic acids synthesized in vitro: the role of pyrophosphate hydrolysis

The free-energy change of phosphodiester bond formation from nucleoside triphosphates is more favorable than with nucleoside diphosphates as substrates. Base-stacking interactions can make significant contributions to both delta G degrees ' values. Pyrophosphate hydrolysis when it accompanies the former reaction dominates all thermodynamic considerations. Three experimental situations are discussed in which high-molecular-weight polynucleotides are synthesized without a strong driving force for covalent bond formation. For one of these, a kinetic scheme is presented which encompasses an early narrow Poisson distribution of chain lengths with ultimate passage to a disperse equilibrium population of chain sizes. Hydrolytic removal of pyrophosphate expands the time scale for this undesirable process by a factor of 10(9), while it enormously elevates the thermodynamic ceiling for the average degrees of polymerization in the other two examples. The electron micrographically revealed broad size population from an early study of partial replication of a T7 DNA template is found to adhere (fortuitously) to a disperse most probable representation. Some possible origins are examined for the branched structures in this product, as well as in a later investigation of replication of this nucleic acid. The achievement of both very high molecular weights and sharply peaked size distributions in polynucleotides synthesized in vitro will require coupling to inorganic pyrophosphatase action as in vivo.

Bacteriophage T7 deoxyribonucleic acid replication in vitro: V. Synthesis of intact chromosomes of bacteriophage T7

Gently lysed extracts of Escherichia coli infected with bacteriophage T7 carry out extensive DNA synthesis in the presence of an exogenous T7 DNA template (Hinkle & Richardson, 1974). The amount of DNA synthesized in the in vitro reaction is two to three times the amount of template DNA added to the reaction. Analysis by zone sedimentation through alkaline sucrose gradients indicates that the products synthesized by extracts prepared from phage-infected E. coli mutants deficient in DNA polymerase I are considerably smaller than intact T7 DNA strands. However, when extracts of phage-infected wild-type E. coli are used in the in vitro reaction, the average molecular weight of the product DNA is increased; up to 50% of the DNA is the same length as intact T7 DNA. Such high molecular weight DNA is also synthesized when extracts of T7-infected E. coli polAl mutants are supplemented with homogeneous E. coli DNA polymerase I. As judged by isopycnic analysis, the product DNA is not covalently attached to the template. The accompanying paper (Masker & Richardson, 1976) describes the biological activity of the high molecular weight DNA synthesized in the in vitro system.

Specificity of RNA chain initiation by bacteriophage T7-induced RNA polymerase

Analysis of the nucleotide sequence at the 5′-triphosphate termini of RNA chains synthesized by T7 RNA polymerase from T7 DNA template indicates that nearly all RNA chains synthesized in this polymerase reaction contain the sequence, pppGpGp. In addition, studies carried out on T7 DNA-dependent 32PP i exchange into ribonucleoside triphosphates suggest that immediately following the guanine residues at the 5′-end of RNA formed in the T7 RNA polymerase reaction, there is one or more adenine residues. These results indicate a high degree of specificity of initiation of RNA synthesis by T7 RNA polymerase.

Studies on bacteriophage T7 DNA synthesis in vitro

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.

Characterization of T7-specific Ribonucleic Acid Polymerase: IV. RESOLUTION OF THE MAJOR IN VITRO TRANSCRIPTS BY GEL ELECTROPHORESIS

Abstract The major in vitro RNA transcripts synthesized by T7 RNA polymerase with T7 and T3 DNA templates have been resolved by electrophoresis on polyacrylamide gels. Six discrete size classes of T7 RNAs are found, designated I to VI. The apparent molecular weights, estimated from their electrophoretic mobilities, span a broad range, from 2 x 105 to 5 x 106. At least two minor RNA species, with molecular weights greater than 5 x 106, are also detected. The six major T7 RNA species are synthesized in approximately equimolar amounts, with the exception of species III, which is made in about twice this amount. It is likely that species III is a mixture of two RNAs (IIIa and IIIb) transcribed from separate regions of the T7 genome. Hence, the six major T7 RNA species are tentatively identified with seven late transcription units on the phage chromosome, which are read with equal efficiences. The six (or seven) transcription products are initiated independently with guanosine triphosphate at the 5' terminus, and are elongated at a rate of 230 nucleotides per s under standard in vitro conditions. When T7 RNA polymerase is used to transcribe T3 DNA, a single RNA transcript is found, with a molecular weight similar to that of T7 RNA species III. This suggests an explanation for the reduced rate of T3 RNA synthesis by T7 RNA polymerase in vitro, and implies that T3 promoter sites read by the T3 RNA polymerase are heterogeneous in nature.

Bacteriophage T7 Deoxyribonucleic Acid Replication in Vitro: REQUIREMENTS FOR DEOXYRIBONUCLEIC ACID SYNTHESIS AND CHARACTERIZATION OF THE PRODUCT

Abstract A cell-free system for the study of bacteriophage T7 DNA replication has been developed. DNA synthesis in vitro requires the four deoxynucleoside triphosphates and an exogenous T7 DNA template. The rate of synthesis is increased 5- to 6-fold by the four ribonucleoside triphosphates. The products of the phage gene 4 and gene 5 (DNA polymerase), but not those of genes 2, 3, and 6, are required for DNA synthesis in vitro. The kinetics of synthesis is linear for 20 to 30 min, and 30 min after the start of the reaction the amount of DNA synthesized is 2 to 3 times the amount of template DNA added to the reaction. The product is not covalently attached to the template. Analysis by zone sedimentation through alkaline sucrose gradients indicates that the products of synthesis are short (11 S) DNA chains, suggesting that the sealing of Okazaki pieces into DNA of high molecular weight is inefficient in this in vitro reaction. Examination of the product in the electron microscope reveals many large, forked molecules, indicating that in some cases large portions of the T7 genome are replicated in vitro.

A preliminary map of the major transcription units read by T7 RNA polymerase on the T7 and T3 bacteriophage chromosomes.

Transcription of T7 DNA by T7 RNA polymerase in vitro gives rise to six major size classes of RNAs comprising seven major T7 RNA species. These RNAs are all read from the r-strand of T7 DNA and are not derived from the early (leftmost on the conventional genetic map) region of the molecule. When artifically shortened T7 DNA templates are transcribed, four (I, II, IIIb, and VI) of the seven species are found to be truncated or deleted. This indicates that all are terminated near the right end of the T7 DNA molecule, probably at a common termination site near 98.5%. (Map positions are all given in terms of percentage of total length measured from the left end of the molecule.) Since the approximate lengths of the transcripts are known, the promotor sites for T7 RNA species I, II, IIIb, and VI are tentatively mapped at 56, 64, 83, and 97% on the T7 chromosome. Only a single major T3 RNA is transcribed by T7 RNA polymerase; analysis of transcripts directed by shortened T3 DNA templates indicates it is analogous to T7 RNA species IIIb. Hence the promotor and terminator sites for T3 species IIIb are tentatively mapped at 83 and 98.5%, respectively, on the T3 chromosome. The major transcripts read by T7 RNA polymerase from T3-T7 hybrid phage DNAs vary, depending on which regions of the T7 chromosome are present. This provides an alternative method of mapping the strong T7 promotor sites on the T7 chromosome.