A T9G mutation in the prototype TATA-Box TCACTATATATAG determines nucleosome formation and synergy with upstream activator sequences in plant promoters

We had earlier reported that mutations to G and C at the seventh and eighth positions in the prototype TATA-box TCACTATATATAG inhibited light-dependent activation of transcription from the promoter. In this study, we characterized mutations at the ninth position of the prototype TATA-box. Substitution of T at the ninth position with G or C enhanced transcription from the promoter in transgenic tobacco (Nicotiana tabacum) plants. The effect of T9G/C mutations was not light dependent, although the 9G/C TATA-box showed synergy with the light-responsive element (lre). However, the 9G/C mutants in the presence of lre failed to respond to phytochromes, sugar, and calcium signaling, in contrast to the prototype TATA-box with lre. The 9G/C mutation shifted the point of initiation of transcription, and transcription activation was dependent upon the type of activating element present upstream. The synergy in activation was noticed with lre and legumin activators but not with rbcS, Pcec, and PR-1a activators. The 9G mutation resulted in a micrococcal nuclease-sensitive region over the TATA-box, suggesting a nucleosome-free region, in contrast to the prototype promoter, which had a distinct nucleosome on the TATA-box. Thus, the transcriptional augmentation with mutation at the ninth position might be because of the loss of a repressive nucleosomal structure on the TATA-box. In agreement with our findings, the promoters containing TATAGATA as identified by genome-wide analysis of Arabidopsis (Arabidopsis thaliana) are not tightly repressed.