MCDB 165A Assessing Protein Function Flashcards

Core Questions:

How do you identify proteins involved in a biological process of interest?

How do you determine the biological function of a protein of interest?

Forward Genetics

Start with a biological process of interest; find genes involved in the process.

Reverse Genetics

Start with a gene; identify its biological function.

Genetic Screens (Forward Genetics)

Identify your biological process of interest, and then purposefully mutagenize the model organism by a chemical. Mutations will be created randomly throughout the genome.

Identify animals deficient in the process of interest. Then sequence/perform genetic linkage experiments.

Genetic Screens- Sevenless Example

One group screened for flies with defective eyes, specifically messed up ommatidia where the R7 photoreceptor was missing. This led them to find the sevenless gene. By screening mutants over years, and through many biochemical/cloning experiments, they were able to determine the signal transduction pathway (sevenless is an RTK receptor).

RNAi/siRNA/dsRNA/shRNA

RNAi is a roughly 20 nucleotide double stranded RNA. Cells have a complex called RISC that separates these RNAi strands and finds a complementary match between the RNAi strand and the mRNA.

RISC then destroys the entire mRNA. So the mRNA never makes the protein (knockdown).

Did the RNAi successfully knock down the protein a researcher wants to get rid of?

Use a western blot or immunofluorescence to determine if a protein has been reduced.

How do you know that an RNAi phenotype is not due to toxicity? Any double stranded RNA could just make the cells sick and die.

You do a control with a nonspecific double stranded RNA. You often use the same sequence of nucleotides and scramble them (so same length and composition). This is called a "scrambled control”.

How do you know that the RNAi phenotype is not due to an off-target effect? The RNAi could knock down the wrong gene that may be similar in sequence.

1) Use a different RNAi sequence to target the same gene at a different location. If they have the same phenotype, it is likely targeting the same gene.

2) Rescue the phenotype with an RNAi-resistant cDNA. If phenotype is rescued, this gives a high probability it is the one target gene that is knocked down.

Morpholinos

Single-stranded antisense oligonucleotides chemically modified to be more stable.

Often targeted to the start codon or a splice site. Morpholinos are thus used to block splicing or translation. Same controls as RNA as described before.

CRISPR/Cas

The best way to knockout a gene. A guide RNA brings Cas9 to a specific genomic sequence, Cas9 cleaves DNA specifically at that site and creates a mutation.

Two Conclusions from Rescue Experiment Controls:

If re-introducing the gene reverts the mutant phenotype to WT, then the phenotype was due to loss of that gene.

If a tagged version rescues, then the tag does not disrupt function.

Structure-Function Experiments

Let's say a Protein X has three domains, domain A, B, and C, and promotes the cell becoming brown and polygenal.

We can make a mutant cell ∆Protein X. We can then make ∆a mutant, ∆b mutant, and ∆c mutant. We see that introducing the ∆c mutant causes the cell to turn green and squiggly. So, domain C is needed for the cell to be brown and polygonal.

Alanine Scanning

Replace each amino acid one by one with an alanine (small and not charged, likely will not mess anything up) and test for function to pinpoint key residues.

This is a structure function experiment.