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 methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, 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 biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.
Ramachandiran
V
V
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Willms
C
C
Kramer
G
G
Hardesty
B
B
D000818:Animals; D002417:Cattle; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D003374:Coumarins / Q000494:pharmacology; D004801:Eosine Yellowish-(YS) / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D009239:N-Formylmethionine / Q000494:pharmacology; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D017510:Protein Folding; D011721:Pyrenes / Q000494:pharmacology; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000494:pharmacology; D013997:Time Factors
D003374:Coumarins; D005456:Fluorescent Dyes; D010455:Peptides; D011721:Pyrenes; D012358:RNA, Transfer, Met; D009239:N-Formylmethionine; C030984:pyrene; C030123:coumarin; D015500:Chloramphenicol O-Acetyltransferase; D013884:Thiosulfate Sulfurtransferase; D004801:Eosine Yellowish-(YS)