2. Predict the protein product and location that would be produced in each of th

Question

2. Predict the protein product and location that would be produced in each of the following conditions employing in vitro translation of preprolactin mRNA to examine the targeting mechanism of secretory proteins to the endoplasmic reticulum. The first three conditions are given as an example. A standard import reaction contains preprolactin mRNA, ribosomes, ER microsomes, cytosol and energy PRODUCT LOCATION Standard prolactin nside microsomes Leave out cytosol preprolact in outside microsomes Leave out microsomes 1st 70 amino acids outside microsomes Cytosol was from cells treated with SRP SiRNA Antibody against SRP receptor was added prior to init iation of translation. Antibody against BIP was added prior to initiation of translation. Translation was initiated without microsomes present After sufficient time for translation, microsomes and an antibody to SRP was added Assume that the ant ibody can only inhibit SRP function from the time of ant ibody addition i.e. it cannot reverse SRP function or binding that occurred prior to antibody addition.

Explanation / Answer

Please find the answers below:

Condition Product Location Reason Cytosol was from cells treated with SSP siRNA No product formation No product formation siRNA would silence the mRNA specific to SSP and hence no translation would take place either in vitro or in vivo Antibody against SRP receptor was added prior to inititaion of translation Prolactin Outside the microsomes An antibody can bind to a receptor only after it has achieved its final functional form i.e. after conversion for pre-prolactin to final biologically active prolactin. Antibody aganst BIP was added prior to inititation of translation Prolactin Outside microsomes The interaction of microsomes with pre-prolactin requires synthesis of biological form of prolactin which is found outside the microsomes Translation was inititated without microsome present. After sufficient time for translation, microsomes and an antibody to SRP was added No product formation No product formation Presence of rough endoplasmic reticulum or aggregated microsomes is essential for carrying out translation, failure of presence of which prevents translation of protein altogether, hence binding of any antibody to the target protein

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