Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle.
Photoreactive moieties were incorporated into nascent polypeptides in a wheat germ protein-synthesizing system by using a plasmid-derived preprolactin mRNA and a Lys-tRNA analog, N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA (epsilon ANB-Lys-tRNA). The presence of the abnormally large amino acid side chains in the nascent chains did not impair function: complete preprolactin chains were synthesized in the absence of the signal recognition particle (SRP), elongation was arrested in the presence of SRP, and SRP-dependent translocation across the membrane of the endoplasmic reticulum and signal peptidase cleavage were observed in the presence of salt-extracted microsomes. Photolysis of elongation-arrested ribosomes resulted in several light- and epsilon ANB-Lys-tRNA-dependent crosslinks. By using antibodies specific for each of the proteins, one covalent complex was shown to be a photocrosslink between the preprolactin nascent chain and the 54-kDa protein subunit of SRP. This demonstrates that the N-terminal end of a secretory protein is located adjacent to the SRP in elongation-arrested ribosomes and strongly suggests that the signal sequence is recognized by and binds to the 54-kDa subunit of SRP. The other photocrosslinks involve as-yet-unidentified proteins in the large ribosomal subunit, indicating that this method of incorporating probes provides a powerful approach to examining the environment and interactions of the nascent chain during translation and translocation across the membrane of the endoplasmic reticulum. The Lys-tRNA analog also successfully photoaffinity-labeled the Escherichia coli elongation factor Tu (EF-Tu) in the epsilon ANB-Lys-tRNA.EF-Tu.GTP ternary complex.
D000345:Affinity Labels / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008027:Light; D008239:Lysine / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D011388:Prolactin / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis*; D021382:Protein Sorting Signals / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism
D000345:Affinity Labels; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012346:RNA, Transfer, Amino Acyl; C017671:preprolactin; D006160:Guanosine Triphosphate; D011388:Prolactin; D010444:Peptide Elongation Factor Tu; D008239:Lysine