Hi. I'm looking in to doing somein vitro transcription from a linear dsDNA template, or a linearised plasmid. When my T7 RNA pol runs along generating the mRNA, once its hits the 3' end of the DNA, will it just fall off and release the mRNA at that point? i.e. if I design my DNA template to end have a particular 3' end, will the mRNAitself have a the exact same (complimentary) 3' sequence only? Knowing the 3' end of the mRNA is quite important for the downstream application I have in mind.
(not sure if it matter but this is bacterial in vitro transcription)
Many thanks!
T7 RNA polymerase is known for adding 1-2 extra ribonucleotides in template-independent way after it ended to transcribe the DNA template. For a DNA template of coding length L, you should expect a mixture of RNA products L, L+1 and L+2. In some special cases, you may run into what's called "turn-around transcription" when T7 RNA pol switches from coding strand to non-coding strand at the end of dsDNA template without dropping the RNA synthesis, and then you get your RNA product much longer than expected. Also, you may get a number of shorter-than-expected RNA products due to premature termination on stable internal hairpins. It's rare to see a homogeneous RNA product in this kind of reactions. Usually you need to run a denaturing PAGE to get rid of all the side products from your raw RNA prep.
See our manuscript (and references cited therein).
https://academic.oup.com/nar/article/46/18/9253/5096072
Also note that we have published the first of several solutions to this problem
Article Efficient inhibition of RNA self-primed extension by additio...
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