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 provided table of codons, and your understanding of the processes of Activation, Initiation, Elongation, and Termination, answer the following questions.  
   1. 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.  
   2. 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?
   3. How is the methionine residue used for prokaryotic initiation of translation unique – draw it.  

Answer 1

ANSWER a) Puromycin acts on both prokaryotic and eukaryotic cells. It has a structure similar to that of the 3′ end of the aminoacyl-tRNA carrier of tyrosine or phenylalanine. For this reason, it occupies ribosomal site A and binds to the polypeptide chain synthesized by peptidyl transferase, blocking the entrance of the next aminoacyl-tRNA. Due to the weak binding of puromycin to ribosomal site A, the formed polypeptide chain is released and protein synthesis is prematurely interrupted,

ANSWER B) Due to different media for bacterial growth that is 14C 15N and 12C 14N media the N groups and C groups incoporated in amino acid have different isotopes thus leading to different peptide ultimately leading to different ribosome structure. There will be two diffrent  band of 70S ribosome. Ribosome of bacteria in 14C 15N media will be lighter comapre to 70S of bacteria grown 14C 15N media.

ANSWER C

The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-tRNAfMet). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.