Question

Laboratory studies of protein synthesis often involve the addition of a radioactively labeled amino acid and natural or synthetic RNAs to cell extracts that provide the ribosomes, translation factors, tRNAs etc necessary for translation. Conversion of the labeled amino acid into protein is measured by precipitation using trichloroacetic acid (only proteins precipitate under these conditions). Conversion of amino acids into an acid-precipitable form is measured as a function of time to estimate the rate of protein formation.In the following experiment, poly(U) is used as a synthetic mRNA in translation experiments carried out with wheat germ cellular extract (wheat germ is a eukaryote). Although the mRNA in this case does not have a Shine-Dalgarno sequence or initiator AUG codon, it is translated into protein.

Answer the following two questions:

(a) What labeled amino acid would you add to the reaction mixture?

(b) What product will result from translation of the poly (U)? For each of the following observations, explain the results. Assume that in a complete system 3000 cpm (counts per minute) are incorporated into an acid-precipitable protein at the end of a 30 minute reaction and that values below 150 cpm are not significantly above the background level.

(c) 85 cpm is precipitated when RNAseA is added to the reaction

(d) 2900 cpm is precipitated when chloramphenicol is added to the reaction

(e) 300 cpm is precipitated when cycloheximide is added to the reaction

(f) 640 cpm is precipitated when puromycin is added to the reaction

(g) 1520 cpm is precipitated when puromycin and extra wheat germ tRNA are added to the reaction

(h) 120 cpm is precipitated when poly(U) is substituted with poly(A) in the reaction

Answer 1

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