Efficient and faithful in vitro translation of natural and synthetic mRNA with human ribosomes.
Efficient systems for in vitro translation are of importance for biochemical and gene expression studies as well as for biotechnological developments. We optimized a cell-free translation system using subcellular fractions from human placenta and high quality placental tRNAs isolated using a simple and fast procedure. The postmitochondrial fraction or a reconstituted system containing soluble proteins plus polysomes were able to efficiently translate endogenous and exogenous mRNAs. Optima for ions, enzymes, tRNA and energy mix components were determined for a poly(U)-directed poly(Phe) synthesis test. The use of homologous tRNAPhe, omission of commercial creatine kinase, and addition of 3.5 mM spermidine at near physiological magnesium concentration (2.5 mM), were the most significant improvements. Under optimal conditions, poly(Phe) synthesis proceeded at a maximal initial rate of 1.2 Phe/80S/min at 37 degrees C, while natural mRNA translation by S-30 started at a near in vivo estimated rate of 0.3-0.5 amino acid/80S/sec. Furthermore, natural mRNA directed the synthesis of a family of polypeptides closely resembling the pattern of cytoplasmic proteins in both, molecular weight and relative amounts. This efficient and faithful system is of interest for biochemical studies of the human translational machinery, as well as a basis for screening new drugs affecting protein synthesis in pathogenic microorganisms.
Ferreras
Ana C
AC
Centro de Investigaciones Biomédicas de la Universidad de Carabobo (BIOMED-UC), Facultad de Ciencias de la Salud, Maracay, Venezuela.
Bandeira
Eduardo
E
Cayama
Edmundo
E
Zambrano
Reina
R
Avila
Horacio
H
Yépez
Antonio
A
Triana
Juana L
JL
Triana-Alonso
Francisco J
FJ
D002474:Cell-Free System; D003402:Creatine Kinase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005821:Genetic Techniques; D006801:Humans; D066298:In Vitro Techniques; D007477:Ions; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010920:Placenta / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D007477:Ions; D010455:Peptides; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D011072:Poly U; D012343:RNA, Transfer; D003402:Creatine Kinase