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Basal Activity of the SP6 Promoter

In vitro experiments are those conducted outside a living organism in a test tube or similar environment. Scientists can make RNA copies or transcripts of a gene in vitro through a process called in vitro transcription. The enzyme that makes the RNA transcript based on the gene is called an RNA polymerase. One such molecule is the SP6 polymerase. It requires a stretch of DNA called the SP6 promoter in order to function properly.

Promoters
- A promoter is the region of DNA bound by an RNA polymerase to initiate transcription. The SP6 polymerase requires that a very specific sequence be present in the promoter in order to bind, so when the SP6 polymerase is used for in vitro transcription, the gene must contain the SP6 promoter for transcription to occur. The SP6 polymerase is from a type of virus called a bacteriophage; biotech companies produce this polymerase by infecting salmonella bacteria with phage and subsequently isolating the polymerase.
Expression
- Basal expression or activity refers to the level of expression in the absence of other molecules that will increase or decrease the level of transcription. Activators or repressors, for example, are proteins that can bind to the promoter or to other gene regulatory regions and either assist in recruiting the RNA polymerase or prevent it from binding. Inducers are molecules that boost production of the gene product by raising expression levels.
SP6
- Basal activity for the SP6 promoter will vary depending on the conditions of your experiment. The most important consideration, of course, is how your experimental conditions can affect activity and how SP6 compares to other systems. In general, SP6 polymerase and promoters deliver lower efficiency than the T7 or T3 in vitro transcription systems, although under ideal conditions SP6 is roughly comparable to these other two. Like SP6, both T7 and T3 use an RNA polymerase from bacteriophage.
Conditions
- Salt concentration and temperature are two important factors in determining basal activity. SP6 is very sensitive to salt concentration, so higher salt concentrations can decrease its activity greatly. All else remaining equal, expression is significantly greater at 104 degrees Fahrenheit than at 99 degrees Fahrenheit. Nucleotide concentration is another important consideration. Low concentrations can potentially cause transcription to terminate prematurely and thereby decrease your efficiency. Increasing the nucleotide concentration will avoid this problem. If you are synthesizing RNA probes, however, adding too much unlabelled nucleotide will decrease the amount of labelled nucleotide in the final product.

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