A possible explanation for polypeptides being about 20 amino acids longer when synthesized by free ribosomes (not attached to the endoplasmic reticulum, ER) relates to the presence and processing of signal sequences. Polypeptides destined for the ER typically have an N-terminal signal peptide of about 20 amino acids that directs the ribosome to the ER membrane. When synthesis occurs on ER-bound ribosomes, this signal peptide is recognized and the nascent chain is co-translationally translocated into the ER lumen, where the signal peptide is cleaved off by signal peptidases, resulting in a shorter mature polypeptide. In contrast, when polypeptides are synthesized by free ribosomes in the cytosol without ER targeting, the signal peptide is not cleaved because the polypeptide does not enter the ER. As a result, the full- length polypeptide including the signal sequence is produced, making it about 20 amino acids longer than the mature form synthesized on ER-bound ribosomes
. This difference in length is due to the absence of co-translational translocation and signal peptide cleavage in free ribosome synthesis, leading to the retention of the signal sequence in the final polypeptide.
