Question

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. What results would you expect to see with 30S ribosomal subunits alone? With 50S subunits alone? With both subunits and all amino acids except leucine, which is required in the 20th position of the polypeptide?

Answer 1

In presence of 30s subunit alone

The initiation complex would be formed but no translation would occur because first f-met-tRNA would transfer amino acid to P site only after binding of 50s subunit.

In presence of 50s Subunit alone

Translation would not start at all because it is the 30s subunit which along with IF1, 2 and 3, mRNA, f-met-tRNA forms initiation complex. Hence no initiation complex will be formed.

If amino acid leucine is absent

The process will take place normally untill 19th amino acid is incorporated after that uncharged tRNA would bind to ribosome which would produce alarmone (p)ppGpp by activation of the RelA enzymein (this process occurs in E.coli). This would stop the translation and Rel A controls the RNA pol activity to reduce the transcription of that particular mRNA. It also enhances production of genes responsible for biosynthesis of leucine.

Once leucine is formed it will be started again by SpoT enzyme which hydrolyzes alarmone molecule.

If leucine not produced the polypeptide and mRNA are degraded.