- 1 Overall goal
- 2 Part 1: Rnt1p
- 3 Part 2: Guide RNAs
- 4 Experimental checkpoints for regulated expression
- 5 Applications for mt gene regulation, in the unlikely case that this system works
Make a tool to regulate gene expression of any mt gene. Tool would have two-parts and be nuclear-encoded.
- Part 1: Rnt1p (RNaseIII enzyme), targetted to mt using sig sequence from HEM1 or COX4.
- Part 2: Guide RNAs, targetted to mt using lysing-tRNA-CUU (tRK1) import system.
Part 1: Rnt1p
Gen'l info about RNases
S. cerevisiae RNAases
Part 2: Guide RNAs
Structural requirements for guide RNAs
Cell components needed for moving guide RNA to mt
- piggy back
Experimental checkpoints for regulated expression
Part 1: Rnt1p in mt
- add tag to gene to follow localization of protein product
- microarray induced/uninduced to look for effect of RNase in mt.
- mtBARNase leads to resp- cells when expressed at low level (YPEG from GALS promoter) and is toxic at high levels (SC-U/Gal), unless simultanously express mtBARSTAR inhibitor protein Mireau, Arnal and Fox in Mol Gen Genomics 2003 270:1-8.