Omar Bennett
SP6 RNA polymerase is a DNA-dependent RNA polymerase that is bacteriophage-specific. It is a stable enzyme that is easily purified and requires a DNA template and Mg2+ ion for RNA synthesis. SP6 polymerase is used for in vitro mRNA synthesis and is highly specific for the SP6 promoter. It is also highly active in the synthesis of poly(rG) with poly(dI) . (dC) as a template. SP6 polymerase is highly sensitive to salt inhibition and is slightly more active at 40°C than at 37°C. The polymerase gene was efficiently expressed in E. coli cells, and its enzymatic properties were similar to those synthesized in SP6 phage-infected Salmonella typhimurium.
| Characteristics | Values |
|---|---|
| SP6 RNA polymerase | A DNA-dependent RNA polymerase |
| Bacteriophage SP6 | A small, virulent bacteriophage that grows on Salmonella typhimurium LT2 |
| SP6 RNA polymerase activity | Resides in a single polypeptide chain of Mr = 96,000 |
| SP6 RNA synthesis requirements | A DNA template and Mg2+ ion |
| SP6 RNA polymerase stimulation | Bovine serum albumin of spermidine |
| SP6 RNA polymerase inhibition | Thiol-reactive reagents |
| SP6 RNA polymerase optimal temperature | 40°C |
| SP6 RNA polymerase maximum salt concentration | 50 mM |
| SP6 RNA polymerase gene expression | Inserted into an E.coli expression vector |
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What You'll Learn
SP6 RNA polymerase is a DNA-dependent RNA polymerase
SP6 polymerase is often used for in vitro mRNA synthesis and is suitable for various applications in research and biotechnology. It is particularly useful for large-scale syntheses due to its preferred start site of 5'GAAGA, which reduces the likelihood of RNA aggregation. SP6 polymerase also consistently produces higher yields of RNA compared to T7 polymerase, and its reactions can be easily scaled up to generate milligram quantities of RNA.
The use of SP6 polymerase extends the range of possible 5' sequences of RNA products. It requires a complete duplex DNA substrate for efficient synthesis, unlike T7 polymerase, which can work with only the 18 base promoter region being double-stranded. SP6 polymerase's efficiency in synthesizing RNA from short double-stranded DNA templates has been demonstrated in various studies, including those by Melton et al. and Kotaoi et al.
Transcription by SP6 RNA polymerase exhibits a unique dependence on ATP, requiring a higher concentration than other nucleotides. This requirement is influenced by the template's topology, as it is partially relieved when the SP6 template is supercoiled. The initiation of transcription by SP6 polymerase may require partial denaturation of the template in the promoter-proximal region, which can be achieved through negative supercoiling or increasing the ATP concentration.
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SP6 is a small, virulent bacteriophage
Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries. One of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.
The SP6 genome is transcribed in a temporally ordered manner, and transcription by SP6RP gives rise to 10 discrete RNA species. This finding suggests that discrete sites present in the SP6 genome serve as specific initiation and termination signals. One terminator sequence for the SP6 species IX RNA transcript has been identified, cloned, and sequenced. This sequence appears to be analogous to the stem-loop structure found at a comparable position in the T7 genome and other rho-independent terminators.
Preliminary studies have provided evidence for the presence of this termination sequence in the SP6 genome, but the termination efficiency has not been fully characterized. SP6 has been reported to be closely related to phages K1-5, K5, and K1E. Scholl et al. reported that SP6 encodes a tail protein that is in the same family as the tail spike protein of the otherwise apparently unrelated phage P22.
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SP6 RNA polymerase is highly active in the synthesis of poly(rG) with poly(dI)
SP6 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific for the SP6 phage promoter. The 98.5 KD polymerase catalyses in vitro RNA synthesis from a cloned DNA template under the SP6 promoter. RNA synthesized using the SP6 RNA polymerase is suitable for many applications in research and biotechnology.
The SP6 RNA polymerase is highly active in the synthesis of poly(rG) with poly(dI). This reaction is unlikely to involve promoter-like sites but appears to reflect the general catalytic capacity of the polymerase. Cleavage of the SP6 RNA polymerase with trypsin, which eliminates SP6-transcribing activity, has little effect on poly(rG) synthesis. This indicates that the catalytic portion of the polymerase can be separated from the RNA polymerase holoenzyme.
The synthesis of RNA by SP6 RNA polymerase requires a DNA template and an Mg2+ ion. It is strongly stimulated by either bovine serum albumin or spermidine. The enzyme is inhibited by thiol-reactive reagents, suggesting the presence of essential sulfhydryl residues. SP6 RNA polymerase is also highly sensitive to salt inhibition, with overall salt concentration not exceeding 50 mM.
SP6 RNA polymerase is slightly more active at 40°C than at 37°C. Incubation at 40°C may be considered for RNA transcripts containing strong secondary structures.
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SP6 RNA polymerase is sensitive to salt inhibition
SP6 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific for the SP6 phage promoter. It is used for in vitro mRNA synthesis and is suitable for many applications in research and biotechnology. SP6 RNA polymerase is also extremely sensitive to salt inhibition.
The SP6 RNA polymerase enzyme is stable and easily purified to homogeneity in good overall yield. It requires a DNA template and Mg2+ ion for RNA synthesis and is strongly stimulated by either bovine serum albumin or spermidine. The enzyme is inhibited by thiol-reactive reagents, indicating the presence of essential sulfhydryl residues. SP6 RNA polymerase possesses a stringent promoter specificity, similar to but distinct from other phage RNA polymerases.
The overall salt concentration for SP6 RNA polymerase reactions should not exceed 50 mM. This is because high salt concentrations can reduce the binding of RNA to the polymerase, which can limit rebinding and reduce dsRNA synthesis.
To increase RNA yield, rNTP concentrations can be raised to 4 mM each, and MgCl2 to 20 mM. SP6 RNA polymerase is slightly more active at 40°C than at 37°C, and incubation at the higher temperature may be considered for RNA transcripts containing strong secondary structures.
SP6 RNA polymerase has been studied by numerous researchers, including Butler, Chamberlin, Melton, Maniatis, Zinn, and Green, who have contributed to the understanding of its properties, synthesis, and applications.
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SP6 RNA polymerase is stable and easily purified
SP6 RNA polymerase is a stable enzyme that can be easily purified to homogeneity in good overall yield. SP6 is a small, virulent bacteriophage that grows on Salmonella typhimurium LT2. It is morphologically similar to Escherichia coli bacteriophage T7 but is genetically distinct.
SP6 RNA polymerase is highly specific for the SP6 promoter and can catalyze in vitro RNA synthesis from a cloned DNA template. The enzyme requires a DNA template and Mg2+ ion for RNA synthesis and is strongly stimulated by either bovine serum albumin or spermidine. Thiol-reactive reagents inhibit the enzyme, indicating the presence of essential sulfhydryl residues.
SP6 RNA polymerase is also highly active in the synthesis of poly(rG) with poly(dI). (dC) as a template. This reaction reflects the general catalytic capacity of the polymerase, as cleavage of the SP6 RNA polymerase with trypsin eliminates SP6-transcribing activity but does not affect poly(rG) synthesis. This suggests that the catalytic portion of the polymerase can be separated from the RNA polymerase holoenzyme.
SP6 RNA polymerase is extremely sensitive to salt inhibition, with an overall salt concentration limit of 50 mM. The enzyme is slightly more active at 40°C than at 37°C, and incubation at the higher temperature may be beneficial for RNA transcripts with strong secondary structures. SP6 polymerase requires a complete duplex DNA substrate for efficient synthesis, and it consistently produces higher yields of RNA compared to T7 polymerase.
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Frequently asked questions
SP6 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific for the SP6 phage promoter. It is used for in vitro mRNA synthesis.
SP6 RNA polymerase requires a DNA template and Mg2+ ion to synthesise RNA. It is strongly stimulated by either bovine serum albumin or spermidine.
SP6 RNA polymerase is used for in vitro mRNA synthesis and is suitable for many applications in research and biotechnology.
SP6 RNA polymerase is slightly more active at 40°C than at 37°C.
