Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation.
The fate of the amino termini of nascent polyalanine, polyserine, and polylysine was monitored by fluorescence techniques as each was translated on Escherichia coli ribosomes. A coumarin probe was placed at the alpha-amino group of a synthetic elongator alanyl-tRNA or a synthetic initiator alanyl-tRNA or at the epsilon-amino group of natural lysyl-tRNA, and each was used to nonenzymatically initiate peptide synthesis. The fluorescent alanyl-tRNAs containing an AAA anticodon were used to initiate polyserine (with a synthetic tRNA(Ser] or polyalanine synthesis from a poly(uridylic acid) template. The fluorescent lysyl-tRNA was used to initiate polylysine synthesis from poly(adenylic acid). Changes in the fluorescence of the amino-terminal coumarin were examined to characterize the environment of the probe as the nascent peptides were extended. Protection from proteolysis and the binding of anti-coumarin antibodies or Fab fragments suggest that the amino terminus of each polypeptide is protected from interaction with proteins (Mr greater than 28,000) until the peptides are extended to an average length of 40-50 residues; however, the fluorescence from the amino terminus of shorter nascent polyalanine and polyserine peptides was readily quenched by methyl viologen (Mr = 257), indicating ribosomes do not shield the nascent peptide from molecules of this size. The data appear to indicate that polyalanine, polyserine, and polylysine are extended from the peptidyl transferase into a protected region of the ribosome such as a groove or tunnel but that this region is readily accessible to small molecules.
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Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
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D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D003374:Coumarins / Q000276:immunology; D019286:Endopeptidase K; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005260:Female; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D010269:Paraquat / Q000737:chemistry; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D014176:Protein Biosynthesis; D012344:RNA, Transfer, Ala / Q000737:chemistry; D011817:Rabbits; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology
D003374:Coumarins; D005456:Fluorescent Dyes; D010455:Peptides; D012344:RNA, Transfer, Ala; C019529:polyalanine; C027794:polyserine; C030861:N-(4-(7-diethylamino 4-methylcoumarin-3-yl)phenyl)iodoacetamide; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D010269:Paraquat