Team:Warwick/Project/Howitworks

There are many elements of the project that must be considered in order to understand the way in which the replicon functions in vivo. The RNA dependent RNA polymerase was the first key element that we needed to get working efficiently in order to maintain the strand in the cell as RNA is inherently unstable. The action of RdRP is depicted below:

The RdRp works to transcribe the RNA strand in both a positive and negative sense, with the 3'UTR and 5'UTR acting as promoters for RdRp action. The siRNA only acts in the negative sense hence the positive strand will not result in cleavage by Dicer, for this reason we attempted to use a stronger promoter at the 5' end in order to produce more funcitonal strands of RNA encoding the active siRNA fragment that will spontaneously form a Dicer recognisable hairpin. Dicer is only present in human cells, therefore the siRNA action could not be tested in Escherichia coli

The action of the siRNA itself can be seen below:

This shows the same diagram as above but focuses on the structure formed in the negative sense RNA strand and the subsequent action of the cellular proteins. The sequence for this hairpin, as designed by a member of our team, can be found at Part:BBa_K1442113

A more detailed diagram of the action of Dicer and RISC during siRNA silencing can be seen below:

One problem we anticipated was the overexpression of siRNA leading to high concentrations of double stranded RNA inside the cell. Double stranded RNA in the cytoplasm is often a sign of viral infection and at a certain concentration this is recognised by the cell and an apoptotic cascade is induced to destroy the cell without dispersal of the viral particles. As our replicon is not transferred between cells this is the last thing we would want to happen. For this reason we installed the MS2 box and associated coat protein. This acts to dampen translation when levels of transcription become too fast and in this way limiting the efficiency of our system so as not to become toxic to the cell. The mechanism of action can be seen below:

The MS2 coat protein is translated at approximately the same rate as the RdRp as it uses the same IRES and is encoded in the same translational unit hence provides an accurate measure of the amount of RdRp present in the cell and can process this information to determine when the system is getting out of hand. THe MS2 coat protein binds to the MS2 box and reduces translation hence reducing the amount of RdRp in the cell and dampens transcription of the strand.

The rest of the elements can be seen on the Parts page.