Activity of U-snRNA genes with modified placement of promoter elements in transfected protoplasts and stably transformed tobacco

In higher plants the promoter elements of pol II- and pol III-transcribed U-snRNA genes are identical, comprising a -30 TATA box and an upstream sequence element, USE. The USE and TATA are centred approximately four and three helical DNA turns apart in pol II and pol III genes, respectively, and it is this difference in the element spacing that determines the RNA polymerase specificity of the gene. In this study we have analyzed the effect of spacing mutations on activity of Arabidopsis U2 and U6 genes in transfected protoplasts of Nicotiana plumbaginifolia and in stably transformed tobacco. In the pol III-transcribed U6 gene the insertions and deletions of either odd or even numbers of half helical turns completely inactivate transcription in transfected protoplasts, consistent with the high conservation of the element spacing found in all plant U-snRNA genes. Surprisingly, while insertions of 50 base pairs (bp) or more into the spacer of the pol II-specific U2 gene inactivate transcription, a deletion of 5 bp or insertions of as much as 20 bp decrease transcription by only 40 to 70%. This relaxed requirement for the conserved element spacing is only seen in transfected protoplasts since the same mutant U2 genes are not transcribed in stably transformed tobacco when transcription takes place from the chromosome. The results provide some clues about possible factor interactions at the promoters of plant U-snRNA genes and also offer an example of major differences in transcription between transiently and stably transformed cells.