N4 and N5

Constructs N4 and N5 as shown by (Fig.1) was designed an made by our advisor, Dr. I. Stansfield, primarily to allow us to characterise the activity of the CUP1 and Gal1 promoters. To do this, addition of the corresponding stimulating chemicals would activate the promoter and induce the production of GFP allowing the quantification of promoter activity. for N4, the inducer was Copper (II) ions. (Cu2+). For N5, the inducer was Galactose.

pRS414

This is one of the constructs that makes up the AyeSwitch as shown by (Fig.2).

The CUP 1 promoter in pRS414 is switched on by the addition of Cu(II) which initiates transcription. The mRNA produced contains a Bbox stem loop that can be bound by N-peptide produced by pRS415. This inhibits the translation of MS2-protein and cyan fluorescent protein, (CFP) which is coded downstream of Bbox stem loop in the pRS414 mRNA.
In the absence of N-peptide, translation of MS2-protein and CFP occurs as usual.

pRS415

This is the other construct that makes up the AyeSwitch as shown by (Fig.3).

The Gal1 promoter in pRS415 is switched on by the addition of Galactose which initiates transcription. The mRNA produced contains two MS2 stem loops that can be bound by MS2-protein produced by pRS414. This inhibits that translation of N-peptide and GFP which is coded downstream of the MS2 stem loops in the pRS415 mRNA.
In the absence of MS2-protein, translation of N-peptide and GFP occurs as usual.

The AyeSwitch

Transforming yeast to contain both pRS414 and pRS415 creates the AyeSwitch.
From (Fig.4) it can be seen that there is mutual inhibition of pRS414 and pRS415 at the translational level. That is because the translated proteins of pRS414 and pRS415 can bind to the corresponding stem loop structures on the opposing construct.

Binding of the stem loops by the correct protein inhibits the movement of ribosomes necessary for translation. This prevents the syntheses of proteins coded downstream of the stem loops in the mRNA sequence.

pMS2

We have used the MET17 promoter to characterise the inhibition of GFP expression caused by the MS2 protein binding to the MS2 loops.

However, with more time we would change this to produce CFP as a replacement for the pRS414 construct.

Ycp lac 22 Fl + CFP

This construct was used during testing to confirm that the CFP sequence being used was functional.