SN1 mapping allows you to see where transciption starts He wanted to show that txn starts a set distance from the TATA box in a class 2 promoter. He got a piece of DNA that had a good class 2 promoter and a piece of DNA that he knew he could cut with restriction endonucleases. The idea is to pair a piece of DNA with label on only one strand. In this case you want the label on the crick strand. You would take this piece of DNA with a hot phosphate on 5 prime end of the crick strand and denature it. The probe DNA helps you see where txn starts. You take that probe DNA and you mix it with transcript thats made by the enzyme. He lets the enzyme make cold mRNA and then mixes the cold mRNA with the probe and then adds S1 nuclease. S1 nuclease will cut anything that is single stranded. Part of the probe is single stranded, so your probe will get smaller. How small your probe will get depends on where transcription starts. This was the procudure that Chambone used to show that RNA polymerase 2 keys on the position of the TATA box. RNA pol 2 doesn’t care where it starts transcribing as long as that transciptional start site is about 25 bp downstream of the TATA box.
Polymerase has no trouble finding the DNA, he noticed that he could do SN1 mapping to show that polymerase when it was given this piece of DNA in normal conidtions it started transcribing at 3 G’s that were very close to each other. The enzyme started transcibing at anyone of the G’s. They were about 25 bp downstream of the TATA box. Chambone started making deletions in the DNA and began to see what happens where the transcription start site occurs. He was able to show was that the enzyme doesn’t care which nucleotide it starts on as long as its on the right position relative to the TATA box. Chambone was able to show that transciption was able to start at different spots. This was due to deletions that he had made between the original start site and the TATA box. The difference was that...
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