Nascent chain, mRNA, and ribosome complexes generated by a pure translation system.
Ribosome display is based on the concept that ternary complexes consisting of a nascent chain, ribosome, and mRNA can be generated, thereby establishing the linkage between genotype and phenotype that is essential for evolutionary experiments. With cell extract-based in vitro translation systems, it has been shown that ternary complexes can be generated by omitting the termination codon from the constructs, which can be stabilized at low temperature in the presence of high Mg2+ concentrations. Using an Escherichia coli-based reconstituted in vitro translation system (PURE system), in which all components necessary for the translation reaction were highly purified and reconstituted, ternary complexes could be generated equally well with a variety of sequences at the 3' end of the RNA, even those with a termination codon. Moreover, the generated complexes were stable at temperatures between 4 and 50 degrees C, and are thus highly stable in contrast to previous assumptions.
Matsuura
Tomoaki
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Yanagida
Hayato
H
Ushioda
Junya
J
Urabe
Itaru
I
Yomo
Tetsuya
T
D002474:Cell-Free System; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D018832:Molecular Chaperones / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*
D018832:Molecular Chaperones; D012329:RNA, Bacterial; D012333:RNA, Messenger; C096099:nascent-polypeptide-associated complex