User:Nkuldell/mtDNA pt3

Overall goal

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.

Foundational info: Summary of natural transcriptional control elements Biswas in PNAS 1990 87:9338 and Biswas and Getz in J Biol Chem 1990 265:19053
Foundational info: works for degradation of mRNAs in S. cerevisiae nucleus Lamontagne and Elela in PLoS One 2007 5:e472

Part 2: Guide RNAs

Cell components needed for moving guide RNA to mt

• piggy back

Experimental checkpoints for regulated expression

Part 1: Rnt1p in mt

• 1. add tag to gene to follow localization of protein product
  2. 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.