The effect of a hydrophobic N-terminal probe on translational pausing of chloramphenicol acetyl transferase and rhodanese

Abstract

The effect on translational pausing of a hydrophobic probe, coumarin, at the N terminus of nascent peptides was investigated. Two different proteins, bacterial chloramphenicol acetyltransferase and bovine rhodanese, were synthesized by coupled transcription/translation in a cell-free system derived from Escherichia coli. Protein synthesis was initiated with N-formyl-Met-tRNA for N-acetyl-S-coumarin-Met-tRNA f. Cotranslational incorporation of the coumarin derivative generated nascent polypeptides with a hydrophobic residue at their N termini. The effect of the two N-terminal groups on the size distribution and quantity of the peptides formed by translational pausing was investigated. The N-terminal coumarin caused an accumulation of nascent chloramphenicol acetyltransferase peptides in the mass range of 3.5-4.0 kDa that reflects a delay in translation at this point. No similar effect on rhodanese pause-site peptides was observed. This effect on translational pausing cannot be explained by either mRNA secondary structure or rare codons and tRNA abundance. It is suggested that the effect of N-terminal coumarin on translational pausing is the result of the interaction of the nascent peptide with components of the large ribosomal subunit along the path it follows between the peptidyl transferase center and the exit site on the distal surface.