1. First Describe a toeprint a
1. First Describe a toeprint assay involving E. coli ribosomalsubunits and a fictious mRNA in a cell-free extract that containsall the factors necessary for translation.
a) What results would you expect to see with 30S ribosomalsubunits alone?
b) With 50S subunits alone?
c) With both subunits and all amino acids except leucine, whichis required in the 20th position of the polypeptide?
Answer:
1.
Toeprint assay helps in the identification of exact location ofthe ribosomal subunits of mRNA. This can help in studying themechanism of translation in detail. The technique involves usingcell lysate, either purified or crude, and then addingcycloheximide. This inhibits the mRNA-ribosomal complex, thusinhibiting the process of translation. This complex is then reversetranscribed to produce cDNA. A radiolabelled primer is used forextension, thus allowing for easier detection of cDNA. Since theribosomal units are bound to the mRNA, reverse transcriptase willstop at this point. A cDNA fragment is produced which is thetoeprint of mRNA.
a.
The 30S subunit (P site) of ribosome is involved in initiatingthe process of translation. It helps in binding the initiator tRNAcarrying the start codon and positioning it on the mRNA. It alsomaintains the translational reading frame. This can help inidentifying the nucleotide sequences required for translationinitiation.
b.
50S ribosomal unit is responsible for peptide bond formation andpeptide chain elongation. It attaches to the 30S ribosomal mRNAcomplex after the initiation step. With only the 50S subunits,translation will not begin.
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