Question

G alpha z (Gaz) is a protein of 355 amino acids in length. It is synthesized in the cytoplasm and has no signal
sequence, nor a start-transfer sequence, nor a nuclear import sequence or patch. After its initial synthesis,
Gaz is bound by another protein called a palmitoyltransferase, which covalently attaches a fatty acid called
palmitate to a cysteine side chain near the N-terminus of Gaz. This lipid modification allows Gaz to
associate with the inner face of the plasma membrane. Now, suppose you discover a mutant form of Gaz, and
when you perform a fractionation of cells expressing this protein, you detect it in the cytoplasm. (In a control
sample, you detect normal Gaz bound to the plasma membrane, as expected.) You decide to try cloning
(isolating) the cDNA that encodes this mutant form of Gaz, and in your lab work you obtain three different
clonal human cDNAs with the following sequences (these strands match the mRNA sequence):

1: cDNA A. 5’- cgctgctgccagaccatgggctgtcggcaagcctcagag [hundreds more bases] -3’
2: cDNA B. 5’- cgctgctgccagaccatgggcggtcggcaaagctcagag [hundreds more bases] -3’
3: cDNA C. 5’- cgctgctgccagaccatgggctgtcggcaaagctcagag [hundreds more bases] -3’

A.) Note that these cDNAs include a small untranslated region upstream of the start codon sequence. Of these
three cDNAs, indicate which one encodes the Gaz mutant of interest, and explain your reasoning precisely.

B.) You go into your lab and engineer a cDNA encoding chimeric Ras protein which has the N-terminal 20 amino acids removed and replaced by the N-terminal region of insulin. It is predicted that this chimeric protein will localize to the inner face of the plasma membrane because it is still containing the C-terminal CAAX motif of Ras, which will be recognized and lipid-modified by farnesyl: protein transferase in the cytoplasm. Why is this assumption incorrect?

((this is all the information that is given, let me know SPECIFICALLY what else I could provide!))

Answer 1

(A)

cDNA or complementary DNA is formed by reverse transcription of mRNA by the enzyme reverse trnascriptase. It has a sequence complementary to the mRNA and similar to the template DNA strand (which acted as a template for mRNA). It contains no introns but does contain the UTRs.

A forms 2 hydrogen bonds with T while G forms 3 hydrogen bonds with C. U is present in mRNA isntead of T.

Gaz protein is attached a palimtoyl group at a cysteine residue.

Cts is encoded by 2 codons - UGC UGU

cDNA would contain TGC or TGT.

1: cDNA A. 5’- cgc tgctgc cagaccatgggc tgt cggcaagcctcagag [hundreds more bases] -3’
2: cDNA B. 5’- cgc tgctgc cagaccatgggcggtcggcaaagctcagag [hundreds more bases] -3’
3: cDNA C. 5’- cgc tgctgc cagaccatgggc tgt cggcaaagctcagag [hundreds more bases] -3’

Out of the 3 cDNA sequences all 3 have TGCTGC.

TGT is present in 1 & 3.

Start codon ATG (marked in bold) is present in all 3.

As mentioned above, cDNA contains UTR at 5' end. Since ATG is start codon UTR is present upstream of it at 5' end.

Thus, these codons would not code for Cys.

Thus, 2 is the mutant cDNA as it does not have a Cys coding codon (TGT) downstream start site.

(B)

Signal sequences are short amino acid sequences which help in targeting a protein to its desired location.

ER signal sequences are present at the N terminal and trigger translocation of proteins carrying it to ER. Proteins entering the ER may further be destined to golgi, remain in ER, lysosomes, plasam membrane, secreted out.

insulin is a secretory protein and thus would have an ER signal sequence which would be cleaved upon its translocation to ER.

Ras is a GTPase protein involved in several signaling pathways. C terminal CAAX sequence allows its post-translational modification (farnesylation).

Ala Ala are aliphatic residues while X represents any C-terminal amino acid (depends on the substrate specificity). CAAX containing proteins are involved in cell proliferation, differentiation, nuclear stability, embryogenesis, spermatogenesis, metabolism, and apoptosis. CAAX containing proteins require a prenylation process before being translocated to plasma membrane or nuclear membrane.

Chimeric Ras protein having N terminal sequence of insulin would now be first translocated to ER. This does not allow the initial prenylation to take place. Thus, the assumption that chimeric Ras may still be localized to plasma membrane maybe wrong.