Question

3. Early experiments aimed at sequencing DNA were designed to mimic approaches used for proteins. Enzymatic end group analysis was a particularly popular method because wide variety of nucleases was available. However, while end group analysis with enzymes like aminopeptidase provided sequence information for the first 20 to 30 amino acids of a protein, end group analysis with nucleases like spleen phosphodiesterase failed to reach even those modest lengths. Briefly explain why nuclease-based end group anaylsis is so limited.

Answer 1

The structure of a protein contains a linear chain of polypeptides, having an N terminal and a C-terminal. The C-terminal of the polypeptide is generally found close to the mRNA sequence on the tRNA during translation. However, the N-terminal is freely available in the growing polypeptide chain. Thus, if an aminopeptidase acts upon this growing chain, it will slowly dissect the peptide bonds from the N-terminal and release the amino acids. Thus, a sequential appearance of amino acids will correspond to the linear organization of the polypeptide.

However on the contrary, the polypeptide is joined to the tRNA by an ester linkage. This bond cannot be cleaved by the enzyme like spleen phosphodiesterase. Hence, an enzyme digest cannot be obtained in this case and no sequence information could be obtained.