Question

1. Protein synthesis requires energy in the form of ATP and GTP. Two high-energy phosphate bonds (from ATP) fuel the formation of one aminoacyl tRNA. Formation of the initiation complex requires energy from one GTP. Delivery of each new tRNA to the Asite requires one GTP. Termination requires the hydrolysis of one GTP.   Based on this information, the table of codons, and your understanding of the processes of Activation, Initiation, Elongation, and Termination, answer the following questions.

1. How many high energy phosphate bonds are required for the translation of a 100 amino acid polypeptide, starting with mRNA, tRNA, amino acid and all the necessary enzymes.
2. A portion of prokaryotic mRNA has the following base pair sequence: 5’ACAUCUAUGCCACGA3’. What would happen if the underlined base was mutated to an A.
3. Puromycin is a drug that is an analog of aminoacyl tRNA. It has an amino group that is capable of forming a peptide bond, but no carboxyl group with which to form another peptide linkage. What would be the effect of adding puromycin to cultures of bacteria engaged in protein synthesis.
4. Ribosomes isolated from bacteria grown in heavy medium (¹⁴C ¹⁵N) and from bacteria grown in light (¹²C ¹⁴N) were added to a cell-free in vitro protein synthesis system. At a later time a sample was removed and 70S ribosomes were analyzed for differing densities. How many 70S ribosome densities were found and why?
5. How is the methionine residue used for prokaryotic initiation of translation unique – draw it.  

Please solve all parts (a - e), please explain and be detailed about the biochemistry! Thank you !

Answer 1

a. For each amino acid incorrporation one amini acyl tRNA will be required. So two molecules of TP will be utilized in this step (Step-1).

Delivery of each tRNA to the A site require one GTP. so for one amino acid one GTP will be utilized. (Step-2).

Initiation requires one GTP and termination requires one GTP .

So for 100 amino acid incorporation, total phosphate groups required will be = 100+100+1+1 =202.

b. The underline is missing in the sequence. So i cant answer this question.

c. Puromycin is an aminonucleoside antibiotic and is obtained from the bacteria Streptomyces alboniger. Puromycin antibiotics has structural similarity with 3; end of aminoacyl tRNA carrier of the amno acid tyrosine or phenylalanine. It binds to the 30S subunit of ribosome and thereby inhibits movement or ranslocation of peptidyl-tRNA. Thus Puromycin cause premature termination in protein synthesis and thereby kills bacteria.

d. Ribosome is made up of Ribosomal proteins and rRNAs. So ribosome isolated from bacteria grown in heavy medium ill have rRNAs and protein made up of heavy Carbon and Nitrogen. The 70S ribosome isolated from normal bacteria will have normal rRNA and protein. So when both types of 70S ribosome will be placed on gradient centrifugation. heavy medium isolated 70S ribosome will appear at the lower position (due to heavy density) and the 70S ribosome isolated from normal medium bacteria will be placed at the higher position due to lower density.

e. The methionine which is used in prokaryotic initiation of translation is N-Formylmethionine. In this methionine a formyl group remain attached to the amino group. In eukaryotes, no suchN-formylated methionine is required, normal methionine is incorporated at the first position of the polypeptide. The structure is as follows: