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

The effect on translational pausing of a hydrophobic probe, coumarin, at th e N terminus of nascent peptides was investigated. Two different proteins, bacterial chloramphenicol acetyltransferase and bovine rhodanese were synth esized by coupled transcription/translation in a cell-free system derived f rom Escherichia cell. Protein synthesis was initiated with N-formyl-Met-tRN A(f) or N-acetyl-S-coumarin-Met-tRNA(f). Cotranslational incorporation of t he coumarin derivative generated nascent polypeptides with a hydrophobic re sidue at their N termini. The effect of the two N-terminal groups on the si ze distribution and quantity of the peptides formed by translational pausin g was investigated. The N-terminal coumarin caused an accumulation of nasce nt 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 o n rhodanese pause-site peptides was observed. This effect on translational pausing cannot be explained by either mRNA secondary structure or rare codo ns and tRNA abundance. It is suggested that the effect of N-terminal coumar in on translational pausing is the result of the interaction of the nascent peptide with components of the large ribosomal subunit along the path it f ollows between the peptidyl transferase center and the exit site on the dis tal surface. (C) 1999 Academic Press.