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Dr Charu S. 

CSIT-NET Dec'14-Section C

This is further to the discussion of the Mol. Genetics and RDT part of the the Section C of the Dec'14 CSIR-NET exam . 

You may also refer to the previous posts...which have covered genetics and the subsequent ones will profile Molecular Biology and RDT. 

84.  Puromycin is an antibiotic used to inhibit protein synthesis. Given below are few statements about the antibiotic.

A.  It enters the E-site of the ribosome where it prevents the action of petidy1 Transferase.

B.  It blocks the translocation process by binding to the translocation factor EF-G.

C.  Puromycin resembles the initiatior tRNA, tRNAif-met and binds exclusively to the P- site

D.  It resembles the aminoacy׀ tRNA and binds to the A-site of the ribosome.

E.  Puromycin inhibits only prokaryotic protein synthesis

F.  Puromycin inhibits both prokaryotic and eukaryotic protein synthesis.

Which of the above statement (s) is/ are true?

1.  A and E       2.  B only         3. D and F        4. C and E

Answer: Puromycin is an aminonucleoside antibiotic. It is derived from the Streptomyces alboniger.

A part of this molecule resembles the 3' end of an aminoacylated tRNA.

 It can enter the A site of the ribosome and serves as an acceptor of the peptidyl chain from peptidyl-tRNA present in the P site. This reaction is catalyzed by peptidyl transferase. Under normal conditions, the tRNA at the A site, then translocates to the P site and a new acceptor tRNA comes to occupy the A site. This cycle is repeated till a stop codon occupies the A site; leading to chain termination.

However when a puromycin accepts a peptidyl chain at the P site, further translocation does not take place.  This results in premature termination of the puromycylated nascent  peptide chain.

Since it specifically inhibits peptidyl transfer, both prokaryotic and eukaryotic protein synthesis is inhibited.

Fungi and Gram negative bacteria are relatively resistant to puromycin due to low permeability of the antibiotic inside the cells.

Note: In molecular biology, the concepts of the basic processes of replication, transcription and translation can be assessed by questions such as these. Antibiotics act at various levels of information transfer from DNA to proteins.  Most act on prokaryotes. Puromycin is effective against both because it affects a step which is universal in prokaryotic and eukaryotic protein synthesis. Cycloheximide acts only on eukaryotic protein synthesis.

An in-depth study of the effect of the various antibiotics should be an important area of focus in your preparation.

86.  The expression of a hypothetical gene was analysed by Northern and western blot hybridizations under control and induced condition. The results are summarized below:

             

Expression of genes can be regulated by:

A. Control at transcription initiation

B.  alternative splicing

C.  control of translation initiation

D. Protein stability

Which of the above regulatory machanisms can explain the observations show in the figures?

1.  Only B                    2.  Only A and B           3. Only B and C           4. A,B,C and D

Answer:

First lets assess the northern blot.  A northern blot gives information about the size and relative quantity of the mRNA or transcription product of the gene.

§       As you observe, mRNA is formed both in control and induced conditions, but more in induced. Therefore the transcript is initiated more times under induced conditions. This indicates control at the level of transcript initiation.

§       Secondly the size of the transcript is smaller under induced conditions. This indicates the transcript is differently spliced under induced conditions compared to control. Therefore gene expression is also being regulated by alternative splicing.  

Now let’s concentrate on the western blot. A western blot gives information about the size and quantity of the polypeptide or translation product.

The polypeptide is being produced only under induced conditions, whereas the transcript was produced under both conditions. 

§       It is observed, that under control conditions the transcript is produced (as clear from the northern blot) , but is not being translated (western blot). This indicates regulation at the level of initiation of translation.

§       On the other hand it is possible that the translation product is formed under control conditions, but is less stable, quickly degraded and not detected in a western blot. Whereas regulation is exercised by increasing the stability of the transcript under induced conditions.