Effects of 3′ Terminus Modifications on mRNA Functional Decay during in Vitro Protein Synthesis

Abstract

The pcnB gene, which encodes the principal poly(A) polymerase of Escherichia coli, promotes 3'-polyadenylation and chemical decay of mRNA. However, there is no evidence that pcnB-mediated mRNA destabilization decreases protein synthesis, suggesting that polyadenylation may enhance translational efficiency. Using in vitro translation by E. coli cell extracts and toeprinting analysis of transcripts encoded by the chloramphenicol acetyltransferase (CAT) and beta-galactosidase genes to investigate this notion, we found no effect of poly(A) tails on protein synthesis. However, we observed that 3'-polyguanylation delayed the chemical decay of CAT mRNA and, even more dramatically, increased the ability of CAT mRNA to produce enzymatically active full-length protein in 30 S E. coli cell fractions. This resulted from interference with the primary mechanism for inactivation of CAT transcript function in cell extracts, which occurred by 3'-exonucleolytic degradation rather than endonucleolytic fragmentation by RNase E. Using bacteriophage T7 RNA polymerase to install poly(G) tails on mRNAs transcribed from polymerase chain reaction-generated DNA templates, we observed sharply increased synthesis of active proteins in vitro in coupled transcription/translation reactions. The ability of poly(G) tails to functionally stabilize transcripts from polymerase chain reaction-generated templates allows proteins encoded by translational open reading frames on genomic DNA or cDNA to be synthesized directly and efficiently in vitro.