Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome

Structurally different fluorescent probes were covalently attached to methi onyl-tRNA(f) and tested for their incorporation into nascent peptides and f ull-length protein using an Escherichia coli cell-free coupled transcriptio n/translation system, Bovine rhodanese and bacterial chloramphenicol acetyl transferase (CAT) were synthesized using derivatives of cascade yellow, eos in, pyrene, or coumarin attached to [S-35]Met-tRNA(f) Al of the probes test ed were incorporated into polypeptides, although less efficiently when comp ared with formyl-methionine. Eosin, the largest of the fluorophores used wi th estimated dimensions of 20 x Ilk caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f ),compared with fMet-tRNA(f), with pyrene having the least and eosin the bi ggest effect. Analysis of the nascent polypeptides showed that the modifica tions at the N terminus affected the rate at which nascent CAT peptides wer e elongated causing accumulation of peptides of about 4 kDa, possibly by st eric hindrance inside the tunnel within the 50 S ribosomal subunit, Fluores cence measurements indicate that the probe at the N terminus of nascent pyr ene-CAT peptides is in a relatively hydrophilic environment. This finding i s in agreement with recent data showing crosslinking of the N terminus of n ascent peptides to nucleotides of the 23 S ribosomal RNA.