Does yeast have a post translation pathway for protein synthesis?If so how does it work? Please start explaining from nucleus transcription to translation in yeast with the promoters or genes that are specific to yeast.

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Reid WolffLv2

15 May 2019

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A student written question: New Belgium Brewery creates its own yeast strains for brewing beer. Some yeast strains secret a toxic protein, which is not detectable until after the brewing process. Therefore the brewery must find a way to detect the yeast that secretes this toxic protein. Because they collaborate with Dr. Wolkow, a yeast-whiz, the brewery decides to hire you to work with Dr. Wolkow to make a cDNA expression library from the toxin-secreting strain. You decide to use a bacterial expression vector since it is easy to work with.

1. What components must the expression vector contain?

a. promoter, translation start sequence, cloned cDNA sequence, DNA polymerase

b. DNA ligase, translation start signal, cloned cDNA sequence, polyA tail

c. Shine-Dalgarno sequence, promoter, cloned cDNA sequence, antibody

d. promoter, Shine-Dalgarno sequence, cloned cDNA sequence, transcription termination sequence

2. Suppose the radioactive 3 antibody did not attach to any of the protein products. What can you conclude from this?

a. the toxic protein product is produced by all the yeast strains

b. the toxic protein product is produced by one or more of the yeast strains

c. the toxic protein product is not produced by any of the yeast

d. the yeast DNA was destroyed when the cells were lysed

3. Why would the brewer want to specifically use an expression vector rather than another type of vector?

a. It is specifically for eukaryotic DNA

b. It is used to produce the protein encoded by a cloned gene

c. It is only used for cloning cDNA sequences

d. They want to produce mRNA encoded by a cloned gene

4. This type of blot, where protein is transferred onto the membrane and is then hybridized to a tagged-antibody is a blot.

a. Southern

b. Northern

c. western

d. southwestern

byzantiumclam279

A search of a sequence database with the sequence of HKLP shows a good match to a yeast protein. You decide to use yeast genetics to identify other gene products involved in the movement of the vesicles. You make a deletion mutant in yeast of the gene for kinesin-like protein and observe a phenotype (the yeast strain is called YKLP?). You then perform a genetic screen for the same phenotype. This identifies two other distinct genes, YKS1 and YKS2. You engineer each of YKS1, YKS2 and YKLP to be expressed in yeast as a GFP fusion. Each fusion rescues the mutated version of the equivalent wildtype gene. Using these tools, you look at targeting of GFP versions of the other two gene products in mutants of YKLP, YKS1 and YKS2, separately (the mutant yeast strains are called YKLP?, YKS1?, and YKS2?, respectively)

Assuming that the proteins act sequentially, show the order in which the gene products act to localise the YKLP to vesicles, explaining your reasoning

coralhound606

Escherichia coli is widely used to produce proteins from otherorganisms. You have isolates a yeast gene that encodes an enzymeand you want to produce this enzyme in E. coli. You suspect thatthe yeast promoter would not work in E. coli. Why?

After replacing the yeast promoter with an E. coli promoter, youcan detect RNA from the yeast gene in in the E. coli culture. Butthe RNA is twice as long as the RNA isolated from this gene inyeast, and the enzyme coded for by the gene does not appear to befunctional. Explain why.