Coupled replication-translation of amplifiable messenger RNA. A cell-free protein synthesis system that mimics viral infection.
Amplifiable messenger RNAs (Wu, Y., Zhang, D. Y., and Kramer, F. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11769-11773) were used as templates in coupled replication-translation reactions. These amplifiable mRNAs contained a preselected messenger sequence embedded within the sequence of MDV-1 RNA, which is a small, naturally occurring template for Q beta replicase. When these recombinant mRNAs were incubated in vitro in reactions that contained both an Escherichia coli cell-free translation system and Q beta replicase, the encoded protein was synthesized more efficiently than in corresponding reactions that did not contain Q beta replicase. Moreover, when coupled replication-translation reactions were carried out in a continuous-flow format (Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Yu., and Alakhov, Yu. B. (1988) Science 242, 1162-1164), the synthesis of biologically active protein continued for a prolonged period. The results suggest that the mechanism of replication and translation in coupled reactions is similar to the mechanism by which Q beta phage genomic RNA is simultaneously replicated and translated in Q beta-infected E. coli: protein synthesis occurs on nascent RNA strands; many more sense strands are synthesized than antisense strands; and the integrity of the messenger sequence is preserved because a relatively small number of antisense strands serve as master templates for the synthesis of new messenger strands.
Ryabova
L
L
Institute of Protein Research, Russian Academy of Sciences, Moscow Region.
Volianik
E
E
Kurnasov
O
O
Spirin
A
A
Wu
Y
Y
Kramer
F R
FR
D002474:Cell-Free System; D066298:In Vitro Techniques; D007700:Kinetics; D014176:Protein Biosynthesis; D011777:Q beta Replicase / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D013698:Templates, Genetic; D014779:Virus Replication
D012333:RNA, Messenger; D011777:Q beta Replicase