T7 RNA pol termination signals? - SUMMARY
bernard at elsie.nci.nih.gov
Fri Jun 7 00:46:48 EST 1996
In article <1996Jun4.050619.20308 at alw.nih.gov>, bernard at elsie.nci.nih.gov
>Bottom line: What are the sequences of phage polymerase
>[Story of problems with in vitro transcription deleted]
Just in case anyone was curious as to the answer...
I received two very helpful e-mail replies to my request.
Nick Theodorakis (ntheo at welchlink.welch.jhu.edu) pointed
me to the work of MJ Chamberlin (UC Berkeley) as a good
entry to phage termination signals. It would appear that
the concensus is a hairpin loop followed by a run of U's.
Nathan O. Siemers (siemers at bms.com) pointed me to Novagen's
pET vectors as a source of a typical T7 termination
sequence. Indeed this 43 bp sequence is predicted to form
a very stable (eg. dG = -19.6 kcal) hairpin loop (at least
7 pairs in the stem - more if G~U pairing is permitted) and
trails off to a run of 6 U's.
...and my template for transcription?
I found a loop with a 5 bp stem and a stability of -1.8 kcal
followed by a run of 5 U's. Enough to make me feel at least
a little suspicious (I do get *some* product). Hopefully I
can trim down the template and boost production.
Thank you very much for your help,
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
"He's like an idiot savant -- minus the savant." -Crow T. Robot, MST3k
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