Every bloody protocol suggests adding in DTT when doing in vitro RNA transcription. Why? The rationale seems to be that the cytoplasm traditionally has a reducing environment but as the only protein we care about is the T7 polymerase, why is this necessary.
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1$\begingroup$ You're right. I just looked up a couple of protocols (an example from Promega ) and it has a final concentration of 10 mM DTT. They don't explain why, however it must be important for it to be included. $\endgroup$– user560Mar 27, 2012 at 22:56
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2 Answers
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A quick search on T7 cysteines gave some clues:
Bacteriophage T7-induced DNA polymerase is composed of a 1: 1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin. Preparation of active subunits in the absence of sulfhydryl reagents indicates the reduced form of thioredoxin is sufficient for formation of the active holoenzyme. The oxidized form of thioredoxin, thioredoxin modified at one active site sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified at both active site sulfhydryls by N-ethylmaleimide, are all inactive, being defective in complex formation with gene 5 protein.
Adler and Modrich, J Biol Chem 258:6956 (1983)
There's a more recent paper (Aguirre et al, Inorganic Chemistry 48:4425 (2009)) that mentions the "the enzyme critical sulfhydryl cysteine groups", but unfortunately I only have access to the abstract.
Update: It seems to be an old finding, rather than a rationale concerning the cytoplasmic redox state. According to Chamberlin and Ring, JBC 248:2235 (1973),
General Requirements-The general requirements for T7 RNA synthesis directed by T7 DNA polymerase are shown in Table I. As expected for a template directed polymerase, RNA synthesis shows an absolute requirement for DNA, the 4 ribonucleoside triphosphates and Mg++.
(no surprises there ;)
The activity of the enzyme is reduced significantly if a sulfhydryl reducing agent such as b-mercapto-ethanol is omitted from the reaction. The addition of 10^-5 M p-hydroxymercuribenzoate to the assay system in the absence of b-mercaptoethanol abolished all activity, indicating that the enzyme contains a sulfhydryl group necessary for activity.
However, if you see the table I, the remaining activity after removing bme is still 74%
There seems to be 7 exposed cysteines (Mukherjee et al, Cell 110:81 (2002)), but I could not find any paper discussing their roles.
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2$\begingroup$ Here's a copy of the paper. Enjoy. cl.ly/101U380b3r261i0w0v1d $\endgroup$– jp89Mar 27, 2012 at 23:49
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$\begingroup$ How strange. I'm looking at the crystal structure (1QLN) and there aren't a lot of cysteines near the active site. Will have to take a look at those papers. $\endgroup$ Mar 28, 2012 at 0:39
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$\begingroup$ @jp89 Thanks for the copy of the paper. I'm not sure though if this is allowed here, since it is breaking copyrights. Anyhow, for now I believe it is still fair use. Regarding to the question, I'd follow citations 59-62 from there (last paragraph on page 2/first on page 3). Cheers. $\endgroup$– AleadamMar 28, 2012 at 1:10
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$\begingroup$ Bacteriophage polymerase is active within the cell, which has a reducing environment. Most protocols of intracell proteins include DTT or glutathione for this reason. Disulfides are mostly for secreted proteins. $\endgroup$– shigetaMar 28, 2012 at 13:16
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$\begingroup$ @Aleadam Yeah, that is the sad case. I honestly don't understand the concept of copyrighting human knowledge. $\endgroup$– jp89Mar 28, 2012 at 15:36
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I always assumed it was because DTT is useful in inactivating ribonucleases (by reducing their disulfides) which are notoriously stable and pervasive. It would be pretty unfortunate to get your RNA synthesized only to have it immediately be destroyed by a contaminating RNase.
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1$\begingroup$ Welcome to Biology.SE! This may be correct, but answers are much more likely to receive a favorable response if they are based on research rather than speculation. Consequently you should include supporting references (primary literature is best). Without that support, your answer is indistinguishable from opinion. This is a good example of how to format references. ——— Please also take the tour and then consult the help pages for additional advice on How to Answer effectively on this site. Thank you! 😊 $\endgroup$– tyersomeOct 5, 2020 at 4:48