Summary: Help T7 in vitro transcription - premature bands?

Ulrike Gausmann ugau at mti-n.uni-jena.de
Mon Jun 8 08:47:28 EST 1998


First of all, thanks to all who tried to solve my problem!

In May I asked:


>Hallo,
>
>I am performing in vitro transcription using T7 RNA polymerase (run-off
>transcription, Ribomax-Kit). My controls work fine, no matter if I
>include CAP analogue or not. Now I subcloned another sequence in frame
>to  my reporter gene and the result is the following:
>
>These RNA samples (with and also without CAP analogue) analyzed with gel
>electrophoresis show degradation (?) or premature termination. Since I
>had RNase contamination before, I know how degraded RNA looks like - the
>gel pattern now seems to contain mostly discrete bands. The size of the
>largest RNA species is exactly what I expect from the fusion gene. I
>decreased the reaction temperature to 30 degrees which did not result
>in more complete transcript. Any ideas?
>
>Now I am looking for T7 termination signals in my construct which should
>be special hairpin structures (am I right?). Does anybody know how  to
>detect these sequences (I do not have access to Unix computers)?
>
>Thank you very much for any help,
>Ulrike

Proposals were:

- increase nucleotide concentration up to 2-4 mM (but decrease UTP to
100-200uM)
- add 7% DMSO into reaction
- try another Polymerase (which means cloning into new vector)
- use of PCR-generated templates for transcription
- prolongation of reaction

and : keep tubes etc RNAse free and make sure that template is totally
linearized


literature about T7 termination:

Jeng, ST, Gardner, JF, Gumport, RI. 1990. Transcription termination by
bacteriophage T7 RNA polymerase at rho-independent terminators. J Biol
Chem
265: 3823-3830.

Jeng, ST, Gardner, JF, Gumport, RI. 1992. Transcription termination in
vitro by bacteriophage T7 RNA polymerase. The role of sequence elements
within and surrounding a rho-independent transcription terminator. J
Biol
Chem 267: 19306-19312.


Also, I found an interesting discussion forum concerning this and other
RNA problems:
http://www.nwfsc.noaa.gov/protocols/methods/RNAMethodsMenu.html
(for my problem see
http://www.nwfsc.noaa.gov/protocols/methods/RNAMethodsDocs/04-1998/4.15.98.1079.html) 


What did I try?
Altering reaction conditions like temperature and NTP conc. did not
help. The same with DMSO (worked even worse).
Since I use Bluescript I could easily test T3 polymerase (Riboprobe,
Promega) which resulted in one band of the expected size (but in the
wrong orientation). Therefore I cloned my sequence into the appropiate
T3 site vector - which resulted in multiple banding I had before.

Finally I realized (see newsgroup) that all of the multiple bands are
premature RNA molecules which are not finished after a 1-hour-reaction.
Adding the same amount of polymerase and the extension of the reaction
up to 3 hours resulted in one band of the expected size!! 


Ulrike


-- 
Dr. U. Gausmann			| D-07743 Jena
Institut fuer Anatomie		| Germany
Anatomie II			| Tel +49 (0)3641 938553
Teichgraben 7			| email ugau at mti-n.uni-jena.de



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