INVIVO AND INVITRO ACTIVITIES OF POINT MUTANTS OF THE BACTERIOPHAGE-T7 RNA-POLYMERASE PROMOTER

Two compatible plasmids were recently reported [Ikeda et al. (1992) Nucleic Acids Res. 20, 2517-2524] that together can be used to determine whether a mutant T7 RNA polymerase promoter is active or inactive in vivo. The first plasmid, pKGP1-1, carries T7 gene 1 (the gene encoding T7 RNA polymerase) ligated to a tac promoter, while the second plasmid, pCM-X#, carries the gene encoding chloramphenicol acetyltranferase (CAT) ligated to potential T7 promoters. If the pCM-X# plasmid carries a potential T7 promoter that can be utilized by T7 RNA polymerase, then CAT is produced from transcripts generated by T7 RNA polymerase from the potential promoter on the pCM-X# plasmid. To determine whether Escherichia coli growth characteristics and chloramphenicol (cam) resistance produced by the plasmids pKGP1-1 and pCM-X# reflect the T7 promoter activity of the possible promoters carried by the pCM-X# plasmids, the in vivo and in vitro strengths of the potential T7 promoters were compared and correlated. In vivo promoter strength was determined by measuring the relative amounts of CAT present in E. coli extracts, while relative in vitro promoter strength was measured in transcription assays. The in vivo and in vitro strengths of 22 point mutants of the consensus T7 promoter were shown to correlate with the growth characteristics and cam resistance conferred to E. coli harboring the plasmid pKGP1-1 and the respective pCM-X# plasmid. Furthermore, the correlation of T7 promoter activity with the presence and/or absence of specific functional groups in the major grooves of the wild-type and mutant promoters suggest the following are necessary for the efficient utilization of a potential T7 promoter: (1) a hydrogen bond acceptor at the N7 position of the G at -11; (2) either a hydrogen bond acceptor and a hydrogen bond donor at the 4-keto group of the T and 6-amino group of the A at -9 or the 5-methyl group at the T at -9; and (3) either hydrophobic interactions at the 5-hydrogen of the C at -7 or a hydrogen bond acceptor at the N7 of the G at -7.