Effects of 3 ' terminus modifications on mRNA functional decay during in vitro protein synthesis

The pcnB gene, which encodes the principal poly(A) polymerase of Escherichi a coil, promotes 3'-polyadenylation and chemical decay of mRNA. However, th ere is no evidence that pcnB-mediated mRNA destabilization decreases protei n synthesis, suggesting that polyadenylation may enhance translational effi ciency. Using in vitro translation by E, coil cell extracts and toeprinting analysis of transcripts encoded by the chloramphenicol acetyltransferase ( CAT) and p-galactosidase genes to investigate this notion, we found no effe ct of poly(A) tails on protein synthesis. However, we observed that 3'-poly guanylation delayed the chemical decay of CAT mRNA and, even more dramatica lly, increased the ability of CAT mRNA to produce enzymatically active full -length protein in 30 S E. coil cell fractions, This resulted from interfer ence with the primary mechanism for inactivation of CAT transcript function in cell extracts, which occurred by 3'-exonucleolytic degradation rather t han endonucleolytic fragmentation by RNase E, Using bacteriophage T7 RNA po lymerase to install poly(G) tails on mRNAs transcribed from polymerase chai n 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 p olymerase chain reaction-generated templates allows proteins encoded by tra nslational open reading frames on genomic DNA or cDNA to be synthesized dir ectly and efficiently in vitro.