26 Jun Bio2B03 Working with proteins tutorial
Question
Bio2B03 Working with proteins tutorial
Data analysis activity.This activity is taken from Lodish et al, 7th edition, p623.
The fact that the processes of translation can be reproduced in vitro allows researchers to design experiments that will elucidate the details of how proteins are translocated to various organelles in the cell. Consider the following experiments done with the secreted protein prolactin and how it gets translocated into the lumen of the ER.
If an mRNA lacks a stop codon to terminate translation the newly synthesized polypeptide remains attached to the ribosome. If mRNAs for prolactin are generated that are of different lengths then polypeptides of different lengths will then be produced.
You have generated a set of mRNAs that encode segments of the N-terminus of prolactin of increasing length, and each mRNA is then translated in vitro by a cytosolic translation extract containing ribosomes, tRNAs, aminoacyl-tRNA synthetases, GTP, and translation initiation and elongation factors. Radiolabeled amino acids are included in the translation mixture so that only the polypeptide encoded by the added mRNA will be labeled. After completion of translation, each reaction mixture was resolved by SDS PAGE, and the labeled polypeptides were identified by autoradiography.
A.The autoradiogram shown below shows the results of the experiment in which each translation reaction was carried out either in the presence (+) or the absence (?) of microsomes. Based on the gel mobility of peptides synthesized in the presence or absence of microsomes, how long must the prolactin nascent chain be in order for the prolactin signal peptide to enter the ER lumen and be cleaved by signal peptidase? (Note that microsomes carry significant quantities of SRP bound to the membranes.) What is band B on the gel? (2 marks)
B
Answer:
B.Given this length, what can you conclude about the conformational state(s) of the nascent prolactin polypeptide when it is cleaved by signal peptidase? The following lengths will be useful for your calculation: the prolactin signal sequence is cleaved after amino acid 31; the channel within the ribosome occupied by a nascent polypeptide is about 150 Å long; a membrane bilayer is about 50 Å thick; in polypeptides with an ?-helical conformation, one residue extends 1.5 Å, whereas in fully extended polypeptides, one residue extends about 3.5 Å. (1 mark)
Answer:
C.In another experiment, each translation reaction was carried out in the presence of microsomes, and then the microsomal membranes and bound ribosomes were separated from free ribosomes and soluble proteins by centrifugation. For each translation reaction, both the total reaction (T) and the microsomal membrane fraction (M) were resolved in adjacent gel lanes. Based on the amounts of labeled polypeptide in the membrane fractions in the autoradiogram depicted below, how long is the prolactin nascent chain when SRP binds to take the peptide to the microsomal translocon? Why are there 2 bands in the 130 M lane (D)? (2 marks)
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