The TRTGn Motif Stabilizes the Transcription Initiation Open Complex

The effect on transcription initiation by the extended −10 motif (5′-TRTGn-3′), positioned upstream of the −10 region, was investigated using a series of base substitution mutations in the α-amylase promoter (amyP). The extended −10 motif, previously referred to as the −16 region, is found frequently in Gram-positive bacterial promoters and several extended −10 promoters from Escherichia coli. The inhibitory effects of the non-productive promoter site (amyP2), which overlaps the upstream region of amyP, were eliminated by mutagenesis of the −35 region and the TRTG motif of amyP2. Removal by mutagenesis of the competitive effects of amyP2 resulted in a reduced dependence of amyP on the TRTG motif. In the absence of the second promoter, mutations in the TRTG motif of amyP destabilized the open complex and prevented the maintenance of open complexes at low temperatures. The open complex half-life was up to 26-fold shorter in the mutant TRTG motif promoters than in the wild-type promoter. We demonstrate that the amyP TRTG motif dramatically stabilizes the open complex intermediate during transcription initiation. Even though the open complex is less stable in the mutant promoters, the region of melted DNA is the same in the wild-type and mutant promoters. However, upon addition of the first three nucleotides, which trap RNAP (RNA polymerase) in a stable initiating complex, the melted DNA region contracts at the 5′-end in a TRTG motif promoter mutant but not at the wild-type promoter, indicating that the motif contributes to maintaining DNA-strand separation.