Variation of in vivo efficiency of the bacteriophage T7 terminator depending on terminator-upstream sequences

Abstract

The 40-kb linear genome of bacteriophage T7 contains only one terminator T phi, for the phage T7 RNA polymerase that is located between genes 10 and 11, Its in vitro termination efficiency has been previously found to vary upon any sequence within a transcription unit [Lee, J. T., Kim, H., Moon, K;., Kim, S., and Kang, C. (1991) Mel. Cells 1, 203-209]. In order to see if this variation takes place also in vivo, termination efficiencies within E. coli cells of the same terminator in the three different transcription units were measured in this study. The three recombinant plasmids, pNNTcat, pSNTcat, and pSSTcat, are different only in promoter-proximal, terminator distal sequence, and/or terminator-proximal upstream sequence. They have shown substantially different in vitro termination efficiencies. In vivo termination efficiencies were measured by two different methods. One method was to compare the expression product levels of a terminator-downstream caf gene (CAT activity) in the presence and absence of the terminator. The other was to compare the RNA levels of terminator-upstream and -downstream DNA. The in vivo termination efficiencies were different in three different plasmids when measured by RNA levels: levels: 97% in pNNTcat, 79% in pSNTcat, and 52% in pSSTcat (the latter tao plasmids showed significantly lower values when measured by protein levels). These in vivo efficiencies were substantially higher than the previously measured in vitro values (77%, 35%, and 17%, respectively). Nevertheless, the order of termination efficiency among the three plasmids was the same within E. call cells as in vitro: pNNTcat > pSNTcat > pSSTcat. Thus, termination efficiency of phage T7 RNA polymerase at the T7 terminator T phi varies upon both terminator-proximal and -distal upstream sequences not only in vitro, but also within E. coli cells.