Cell-free translation of calf type III collagen. Effect of magnesium on ribosome movement during elongation.
Calf aortic smooth muscle cell cultures produce both type III and type I collagen. Polyadenylated mRNA species purified from these cells direct the synthesis of prepro-alpha 1(III), prepro-alpha 1(I), and prepro-alpha 2(I) in a rabbit reticulocyte cell-free system. These polypeptides were identified by specific immunoprecipitation, cyanogen bromide peptide mapping, and bacterial collagenase digestion. Lower molecular weight collagenase susceptible polypeptides were also produced in translation reactions incubated under conditions optimized for incorporation of radiolabeled amino acids. Their presence did not appear to result from ribonuclease or protease involvement or from premature termination. Increasing the Mg2+ concentration in the translation system significantly reduced the production of these lower molecular weight species. Pulse-chase experiments indicate that the time required for completion of full length preprocollagen at the high Mg2+ concentration is greatly decreased compared to the low concentration. Additional experiments suggest that the incomplete collagen polypeptides result from pausing of ribosome movement during elongation. The relative synthesis of type III and type I chains was examined as a function of mRNA concentration in the cell-free system. At levels of RNA above saturation, the relative production of type III decreased with respect to type I. These data suggest that the ability of the alpha 1(III) mRNA to initiate translation is less efficient than the mRNAs of alpha 1(I) and alpha 2(I).
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002417:Cattle; D002474:Cell-Free System; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D012333:RNA, Messenger; D003094:Collagen; D008274:Magnesium