Nascent polypeptide chains within the ribosomal tunnel analyzed by cryo-EM

Abstract

The ribosome is a large macromolecular particle that synthesizes polypeptide chains from the substituent amino acid building blocks. The active site for peptide bond formation, the so-called peptidyl transferase center (PTC), is located in a cleft on the intersubunit side of the large ribosomal subunit (reviewed by (Polacek and Mankin, 2005; Simonovic and Steitz, 2009)). As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large subunit and emerges at the solvent side where protein folding occurs. The first hints for the presence of a ribosomal tunnel in the large subunit came from proteolysis protection and immuno-electron microscopy (EM) studies: Using IgG antibodies raised against β-galactosidase or the rubisco small subunit, Lake and coworkers could show that polypeptide chains emerge on the back of large subunit of the bacterial (Escherichia coli) 70S and eukaryotic (plant) 80S ribosome, respectively — some 75 A from the intersubunit interface (Bernabeu and Lake, 1982; Bernabeu et al., 1983). This distance was consistent with the earlier findings that 30–40 C-terminal amino acids of nascent polypeptide chains are protected by eukaryotic and bacterial ribosomes from proteolysis (Malkin and Rich, 1967; Blobel and Sabatini, 1970; Smith et al., 1978).