THE INTERACTION OF BASE-ANALOG SUBSTITUTED LAMBDA P(,R) PROMOTERS WITH ESCHERICHIA COLI RNA POLYMERASE (DNA SYNTHESIS)

Transcription in E. coli is catalyzed by DNA dependent RNA polymerase. This enzyme specifically recognizes the promoter, the DNA sequence at which transcription is initiated. Two regions of highly conserved sequence are located approximately 33 and 10 base pairs away from the site of transcription initiation (+1). These sequences are TTGACA near -35 and TATAAT near -10. A promoter for E. coli RNA polymerase, the rightward promoter of bacteriophage lambda ((lamda)P(,R)), was synthesized by a combination of chemical and enzymatic methods. Site-specific base changes were introduced into the synthetic promoter at regions thought to be important in the specific recognition of E. coli promoters by polymerase. Most base modifications were found to have negligible effect on the activity of the promoter. A few changes at positions of highly conserved sequence in E. coli promoters were found to reduce promoter activity severely or to yield promoters of intermediate strength.Two methyl groups in the -35 region appear to have a composite effect on the function of the promoter. Removal of both of them inactivates the promoter, while removal of either one creates a promoter showing slower reaction kinetics with RNA polymerase. Interaction of polymerase with a strongly conserved T(.)A base pair in the last position of the -10 homology seems to be taking place in the major groove. These results are discussed in relation to current knowledge of E. coli RNA polymerase function.Removal of both methyl groups from the thymidines at the first two positions of the -35 homology by introduction of two uridines inactivated the promoter. This result was further investigated by making the corresponding single modifications in the -35 region. The two promoters having the sequence TUGACA or UTGACA in the -35 homology were functional but displayed slower reaction kinetics than the unmodified promoter. In the -10 region replacement of the T(.)A base pair at -7 with a C(.)I base pair also inactivated the promoter. The only difference between the C(.)I and the wild type T(.)A base pair is found in the major groove. This result suggests that interaction with RNA polymerase at this position occurs primarily in the major groove.