Sunday, July 08, 2007

Trying to make T7 RNAP behave like bacterial RNAP

I have been using coupled in vitro transcription/translation to test whether the secondary structure at the 5' end of sxy transcript blocks translation. Our strong genetic and in vivo results suggest that the sxy-1 mutation weakens 2º structure and so facilitates ribosome binding; the sxy-7 mutation does the opposite.

My in vitro results show a very little bit more translation in sxy-1 relative to wildtype, and about half as much translation with sxy-7. This trend is nowhere near as dramatic as the in vivo results, so Rosie and I have been troubleshooting the in vitro assay in an attempt to make the test tube more like in vivo conditions. A central complication is that the the in vitro assay uses the phage T7 RNA polymerase to achieve high levels of transcription. T7 RNAP is a very fast and processive (ie. it never drops the ball) enzyme. T7 RNAP transcribes about 10 times faster than E. coli RNAP, and this high-speed can result in miss-folding of RNA (Lewicki et al. (1993) showed this by using T7 RNAP to transcribe the 23S rRNA gene: the T7 product was non-functional, but melting and renaturation restored 23S functionality). Yesterday, I repeated the in vitro experiments at 25ºC because Lwicki found that this low temperature slowed T7 RNAP to a speed similar to E. coli RNAP.

We were also concerned that using circular templates for transciption might result in poly-cistronic transcripts; in other words, T7 RNAP may continue to transcribe around the plasmid template and never stop, resulting in a potentially miss-folded string of sxy genes. To test whether this is a problem, I linearized some of my templates before transcription/translation. I will have the results by Thursday, and our modified proceedures may yield results more like our in vivo data.

2 Comments:

At 3:36 PM, Blogger LindsayAWilson said...

Do you have any controls of transcripts that you know should be translated relatively well/badly, and looked to see if their efficiency is dramatically altered in your experimental system?

 
At 1:11 AM, Blogger Layne Adams said...

Hello there, I was searching about t7 and I came across your blog, very informative and entertaining, it shows that your an expert in your field.
I will definitely be back for more. Keep it up!
Cheers!

 

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