Assembly of procollagen mRNA translation products into pepsin-resistant structures.
Proteolytic digestion was used to probe the conformation of the prepro alpha chains synthesized by a mRNA-dependent reticulocyte lysate from chicken calvarial RNA. Pepsin-resistant alpha 1- and alpha 2-like chains were recovered even from translation reactions that were not preincubated below the reported Tm of the unhydroxylated triple helix. The pepsin-resistant structures were stable to thermal denaturation at 45 degrees C and a fraction remained resistant to peptic digestion at 30 degrees C. Interchain disulfide bonds did not appear to be required for the formation or thermal stability of these structures. Pepsin resistance is normally interpreted as evidence for a triple-helical conformation. Therefore, these results suggest that the in vitro synthesized prepro alpha chains contain the requisite information to associate in register for correct helix folding. The unusual thermal stability of these structures is not understood, but this may indicate assembly into higher orders of structure.
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism; D002642:Chick Embryo; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006358:Hot Temperature; D006868:Hydrolysis; D006909:Hydroxyproline / Q000378:metabolism; D010434:Pepsin A / Q000494:pharmacology*; D011347:Procollagen / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis*
D011347:Procollagen; D012333:RNA, Messenger; D010434:Pepsin A; D006909:Hydroxyproline