Effect of tRNA pool balance on rate and uniformity of elongation during translation of fibroin mRNA in a reticulocyte cell-free system.
Unsuccessful attempts to synthesize complete fibroin chains in vitro were previously made in heterologous cell-free system [3]. In the present work, we succeeded to obtain complete translation of purified fibroin mRNA in a rabbit reticulocyte lysate. Whilst this work was being completed [1], similar results were published by Lizardi et al. [4]. The synthesis of full-sized molecules of fibroin (M.W. 360,000) was achieved by adding tRNA from the posterior silk gland to the cell-free system. With tRNA from other sources, both the translation rate and the amount of complete fibroin chains dropped. This effect of tRNA is situated at the elongation levels. Analysis of cell-free synthesized products by polyacrylamide gel electrophoresis shows that smaller discrete polypeptides are accumulated after 120 minutes of incubation. These polypeptides correspond to growing fibroin chains. This pattern of translation products suggests that elongation might decelerate at specific sites of the fibroin mRNA. These results show that a tRNA pool adjusted to mRNA codon frequency is required to obtain the maximal average elongation rate. A stochastic model based on random acceptance of tRNA at the ribosomal A site for the codon-anticodon recognition process can explain this phenomenon. It can also explain the occurrence of the unfinished discrete fibroin polypeptides during in vitro translation.
D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D005349:Fibroins / Q000096:biosynthesis*; D066298:In Vitro Techniques; D010441:Peptide Chain Elongation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D012333:RNA, Messenger; D005349:Fibroins; D012343:RNA, Transfer