T7 promoter contacts essential for promoter activity in vivo

Abstract

T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+1) and extends from -17 to +6. To determine the bases within the T7 consensus promoter that are essential for promoter function a library of mutant T7 promoters was constructed, and the in vivo activity of the mutant promoters was correlated to their sequence. The library of mutant promoters was created by randomly mutagenizing the T7 phi 10 promoter between positions -22 and +6 during the synthesis of oligonucleotides containing the phi 10 promoter. The mutagenized oligonucleotides were then ligated to a promoterless chloramphenicol acetyl transferase gene creating a plasmid (pCM-X#) that can potentially express chloramphenicol acetyl transferase in the presence of T7 RNA polymerase. E. coli containing pCM-X# and a second compatible plasmid carrying T7 gene 1 (T7 RNA polymerase) were screened for chloramphenicol resistance or chloramphenicol sensitivity. The point mutations that were found to inactivate a T7 promoter are a C to A or G substitution at -7, a T to A substitution at -8, a C to A, T, or G substitution at -9, and a G to T substitution at -11.