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Lab Meeting Presentation #1

KS

Kateland Simmons

Transcript

Detecting Yeast tRNA Nucleotidyltransferase

Protein:Protein Interactions Using the Yeast

Two-Hybrid System

What's Next

  • Yeast transformation this week

  • E. coli transformation of MRT4 and GIS2 in the near future

ARP6 Focus

ARP6 PCR Product

ARP6

ARP6 Transformation into E.coli

1 2 3 P N

1500bp

1000bp

5000bp

1500bp

  • Expected product size between 1000bp-1500bp since ARP6 is 1317bp in size.

500bp

  • Colony 2- ARP6 inserted in correct orientation
  • Obtained two fragments after digesting with EcoRV:

--2,250bp and 4,767bp

Ligation of ARP6

Correct Orientation

Wrong Orientation

  • Fragments of 2,250bp and 4,767bp
  • Fragments of 1,850bp and 5,167bp

Potential Candidates

ARP6

LOS1

  • Function: Actin-related protein that binds nucleosomes

  • Size: 1317bp

  • Potential: Identified physical interaction with CCA1
  • Function: Nuclear pore protein involved in

--nuclear export of pre-tRNA

--re-export of mature tRNAs

  • Size: 3303bp

  • Potential: Identified genetic interaction

LRP1

ENO2

  • Function: component of glycolysis and gluconeogenesis

--Also assists in importing tRNA into the mitochondria

  • Size: 1314bp

  • Potential: Identified physical interaction with CCA1
  • Function: Nuclear exosome-associated nucleic acid binding protein; involved in RNA processing, transport, etc.

  • Size: 555bp

  • Potential: Identified genetic interaction

Why is this important?

  • Positive interactions are important for localization of tRNA-NT

  • Helps us see effects of selected proteins on tRNA-NT

Overview

  • Using the yeast two-hybrid system to detect protein:protein interactions between bait (CCA1) and selected proteins.

  • Confirming positive interactions found by high-throughput methods

Positive interaction= transcription will occur

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