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In the experiment, a plasmid called pSA850 was used to investigate the transcription affects. This plasmid is comprised of multiple promoters that are followed by a transcription terminator that yields sequences in different lengths. Some promoters that were present on the plasmid included lacP, galP1 and galP2, pP, bP, and rP. In order to prepare the topoisomers of the plasmid pSA850, it was incubated in a mixture of calf thymus topoisomerase 1 and ethidium bromide. The reactions finished and proteins as well as the ethidium bromide were taken out by a phenol-chloroform extraction and then isoamyl alcohol was used twice afterwards to also extract. TAE-buffered agarose gel containing chloroquine was used to measure the mean linking number difference of the topoisomers that were produced. The linking number was determined to be 332 using the 10.5 base pair per turn and the superhelical densities were also concluded.
In vitro transcription was started in a blend comprising of DNA template, RNAP, Tris-acetate, pH 7.5, magnesium acetate, potassium glutamate, and rRNasin. The beginning blend was incubated for about five minutes and then added to a mixture of NTP containing ATP, GTP, CTP, UTP and [α-32P] UTP in order for the reactions to begin and then incubated. The reactions were then terminated using BRL which is a STOP solution. The mixtures were warmed for two minutes and in order to observe the resulting transcripts and the abandoned molecules that were prepared from the promoters, 3 microliters of each sample was put aboard polyacrylamide-urea sequencing gels. Gene regulatory proteins were present in varying degrees and therefore could determine whether different levels of topoisomers showed different effects.
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Topoisomers ranged in different levels of superhelicity by changing the amount of ethidium bromide concentrations. They were then used as DNA templates to study supercoiling reliance of the promoters in pSA850. Each promoter was experimented with two different amounts of superhelicity and then observed to find the relative transcription of each. Negative supercoiling was determined to vary amid different promoters. Transcription was initiated in some promoters by superhelicity in the DNA and had a different peak of superhealical density when compared to other promoters that showed a different amount or did not show any. A counter association was observed among unsuccessful and full-length RNA production when supercoiling was increased. As the amounts of failed RNA assembly from the promoters decreased, full-length RNA rose which then shows that DNA superhelicity affects promoters and whether they are authorized to continue. Supercoiling can control whether or not some genes are expressed therefore, supercoiling is very important and needs to be set at a specific level in order to affect the promoter in the correct process.