Question

1. First Describe a toeprint assay involving E. coli ribosomal subunits and a fictious mRNA in a cell-free extract that contains all the factors necessary for translation.

a) What results would you expect to see with 30S ribosomal subunits alone?

b) With 50S subunits alone?

c) With both subunits and all amino acids except leucine, which is required in the 20th position of the polypeptide?

Answer 1

1.

Toeprint assay helps in the identification of exact location of the ribosomal subunits of mRNA. This can help in studying the mechanism of translation in detail. The technique involves using cell lysate, either purified or crude, and then adding cycloheximide. This inhibits the mRNA-ribosomal complex, thus inhibiting the process of translation. This complex is then reverse transcribed to produce cDNA. A radiolabelled primer is used for extension, thus allowing for easier detection of cDNA. Since the ribosomal units are bound to the mRNA, reverse transcriptase will stop at this point. A cDNA fragment is produced which is the toeprint of mRNA.

a.

The 30S subunit (P site) of ribosome is involved in initiating the process of translation. It helps in binding the initiator tRNA carrying the start codon and positioning it on the mRNA. It also maintains the translational reading frame. This can help in identifying the nucleotide sequences required for translation initiation.

b.

50S ribosomal unit is responsible for peptide bond formation and peptide chain elongation. It attaches to the 30S ribosomal mRNA complex after the initiation step. With only the 50S subunits, translation will not begin.