BIS183 Lecture 16 - Protein-Protein Interactions II + Large Scale Genetic Analysis

Waht are some examples of membrane bound proteins

• Important in all biological processes/disease

  • Anchored in membrane

  • can have intra and extra cellular parts

  • difficult to purify if membrane bound

  • can’t go near nucleus

• Chemoattractant receptors (G protein-coupled receptors) → ex: leukocytes → bind to invader

• Receptor tyrosine kinases

• Plant receptor like kinases

Describe the methodology of Split Ubiquitin Two Hybrid

Ubiquitin

• Small peptide tag

• target protein for proteolytic degradation

• 26S proteome

Bait: C-terminus ubiquitin + PLV TF (hybrid - split ubiquitin in half)

• Protein of interest + C-term Ubiquitin + PLV TF

Prey: Protein Y + N-terminus ubiquitin

Steps

1) Bait localizes to a membrane outside nucleus

2) Protease cleaves complex due to reconstituted ubiquitin tag which releases PLVTF

3) PLV TF enters nucleus and binds to promoter and can see if transcription occurs

Describe the different transcription results of Split Ubiquitin two Hybrid

1) Autoactivation

• HIS3 - no growth

• LACZ - white

2) Negative control (no bait or prey)

• HIS3 - no growth

• LACZ - white

3) BAIT + PREY - interaction

• HIS3 - +++ growth

• LACZ - blue

4) BAIT + PREY - NO interaction

• HIS3 - no growth

• LACZ - white

What are the differences between a Yeast 2 hybrid and Ubiquitin assays

Yeast 2 Hybrid

• Look at interaction of cytosolic proteins

Ubiquitin

• Look at interaction of Membrane-bound proteins

What can result in false positives and negative in split ubiquitin assays?

False positive

• Interaction conditions may not be typical for bait + prey

• ex: two proteins may never be in the same cell at the same time

False negative

• interaction conditions are not favorable within a cell

• ex: pH, no cofactors, redox

Proof of Principle for transcription factors and how it relates to mutants

Why is it important to have diverse alleles?

• Mutant alleles can be used to dissect transcriptional regulation

Proteins

• Mutation in DNA Binding Domain

  • abolish binding

  • Increase affinity for binding

• Interaction with RAN polymerase

  • Increase or decrease transcription rate

• Domains that interact with co factors

  • Abolish interaction

  • take place all the time

Cis Regulatory Elements

• Destroy the TF binding site in a promoter or enhancer

• Introduce cope of this CRE in a new palce

• Repeat elements - recreate chromatin architecture

What is large scale genetic analysis? How can it be used to determine gene function?

• Bacterial and yeast whole-genome knockout collections

• Systematic and comprehensive mutation of every single gene in a genome

• Can be done independently

• 10s of thousands of mutant lines - many mutant alleles per gene, know where every gene is → target a mutation to that gene

Unbiased

• Randomly mutate the genome

• tracking phenotypes you can determine when you have reached saturations

• multiple alleles at each gene

• “map based” cloning to identify location of mutation

Why is genome assembly and annotation difficult?

• increase in genome size = increase in transposons/repetitive seqs = decrease in open reading frames

• Repeats confound genome assembly from shotgun sequence

• Defining genes is hard (especially non-coding elements)

• target/reverse genetic approaches complements forward/unbiased gene approach

Describe the genome0wide mutant collection done on S. cerivisiae

• 5916 genes knocked out (haploids and heterozygous diploids)

• 1159 essential genes required for life

• Initial testing in common growth conditions

• 673 mutants with altered cell shape

Define Haploid and diploid and hwo they are involved in genome-wide studies

• Haploid - single genome copy, single allele copy phenotype

• diploid - two genome copies, effect of one allele on another

• yeast - haploid-diploid states

• Explore function of the gene by virtue of their mutant phenotype

Diploids + Mendelian Genetics

• Model organisms → self fertilize and cross

• ex: 1 Aa → 1 AA, 2 Aa, 1 aa

What is the methodology for Homologous Recombination-Based Mutagenesis

• Done in yeast - short identical sequences at the start adn end of gene → requries prior knowledge of gene sequences

Complex

• Barcode - short DNA sequences - recognize/tag another sequence

• P - common primer

• Reporter - GFP or HIS3 or LACZ

Leads to gene of interest being deleted

How do we know this is a mutation in our gene of interest?

• PCR → Illumina seq

• Barcodes also help us tell which gene was mutated

Signature tagged mutagenesis

1) Every deletion mutant contains a unique barcode

2)Grow all mutants in a mixed populations and quantify growth

3) Amplify barcode, sequences everything to find mutations that cause slow growth