BIO 2B03 Working with Proteins

| September 28, 2018

Data analysis activity.This activity is taken from Lodish et al, 7th edition,
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.
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)



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)

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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