Differential translation efficiency explains discoordinate expression of the galactose operon

We have used an mRNA-dependent E. coli S-30 translation system to compare the translation efficiencies of two polycistronic transcripts of the galactose operon, the CRP-cAMP-dependent mRNA (P1) and the CRP-cAMP-independent mRNA (P2). The RNAs were prepared in vitro, quantitated by hybridization or gel analysis and translated in a cell free system. The specific protein products were measured, and their quantities were compared with the amount of input mRNA. Our results show that the P2 mRNA synthesizes epimerase, the 5'-proximal gene product of the gal operon, four times more efficiently than the P1 mRNA. The 5'-distal gene products, transferase and kinase, are translated with the same efficiency from both transcripts. Thus the ratio of epimerase to kinase synthesis is four times higher for the P2 mRNA than for the P1 mRNA. This change in epimerase to kinase ratio is identical to that observed in vivo when the cellular cAMP level falls and gal transcription is believed to switch from P1 to P2. We suggest that it is the differential translation efficiency of the epimerase gene on the two different gal transcripts that accounts for this discoordinate expression. Moreover, since the P2 mRNA differs from the P1 mRNA only by the addition of five nucleotides at the 5' terminus and these nucleotides are outside the ribosome binding region we determine for epimerase, the selective difference in the translation efficiency of epimerase is probably mediated by RNA conformation.