Functional domains of a T-DNA promoter active in crown gall tumors

Promoter domains required for transcriptional expression of the 780 gene of T-right (pTi15955) were identified by deletion mutagenesis. Accurate quantitation of transcriptional activity of a series of 5' and internal deletion mutants was achieved by using a double gene vector containing a reference 780 gene as an internal standard. Results of the 5' deletions delineated an activator element located between -440 and -229 base pairs (bp) from the start of transcription. Removal of this region resulted in a 100-fold decrease in promoter activity. Two relatively small internal deletion/substitution mutations at positions -74 to -76 and -98 to -112 reduced promoter activity to 38 and 42%, respectively. In most cases large-scale internal deletions (38 to 151 bp) occurring in various locations from positions -12 to -348 bp caused a significant loss in major promoter activity. However, three internal deletions starting at position -37 and extending upstream as far as -153 bp either had little effect on transcriptional activity or resulted in increased activity. Removal of the TATA motif drastically reduced promoter activity to less than 0.1% of the wild type. A minor start of transcription was detected 60 bases upstream from the major transcriptional start site. This minor promoter shares the same activator element as the major promoter for full activity. Deletion and insertion mutations downstream of the minor promoter TATA demonstrated the role of the TATA box in positioning the start of transcription.