Abstract
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.
Highlights
We have synthesized coumarin maleimide-S-acetyl-MettRNAf to incorporate a fluorescent probe at the N terminus of polypeptides during their synthesis in a cell-free coupled transcription/translation system derived from Escherichia coli (1– 4)
Do the fluorophores at the N terminus affect the amount and the rate at which polypeptides are formed after they are initiated? Are they incorporated into native full-length protein? The last two questions imply that the bulky modification at the N terminus may hinder the required folding of the nascent peptide to first pass through the tunnel inside the large ribosomal subunit (7), to acquire the three-dimensional structure of the native protein
Incorporation of Coumarin, Cascade Yellow, Eosin, and Pyrene Derivatives of Methionine at the N Terminus of Polypeptides—Coupled transcription/translation in the in vitro E. coli system provides a method by which a modified methionine can be incorporated from derivatized Met-tRNAf into a nascent peptide
Summary
We have synthesized coumarin maleimide-S-acetyl-MettRNAf to incorporate a fluorescent probe at the N terminus of polypeptides during their synthesis in a cell-free coupled transcription/translation system derived from Escherichia coli (1– 4). The last two questions imply that the bulky modification at the N terminus may hinder the required folding of the nascent peptide to first pass through the tunnel inside the large ribosomal subunit (7), to acquire the three-dimensional structure of the native protein. We report the effect on translation of three additional N-terminal fluorophores (pyrene, cascade yellow, eosin), which vary in size and structure compared with coumarin. Synthesis of bovine rhodanese (RHO, a thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) or bacterial CAT (EC 2.3.1.28) was initiated in the presence of fluorophore-Met-tRNAf. Formation of nascent peptides and full-length product was analyzed and compared with polypeptides formed with N-formyl-methionine at the N terminus. The results indicate that RHO and CAT could be produced when pyrene-Met or cascade yellow-Met were at the N terminus of the protein; using eosin-Met-tRNAf, the translational machinery worked less efficiently. The results are discussed under the aspect of folding of the nascent polypeptides on the ribosomes
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