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-transcri bed U-snRNA genes are identical, comprising a -30 TATA box and an upst ream sequence element, USE. The USE and TATA are centred approximately four and three helical DNA turns apart in pol II and pol III genes, r espectively, and it is this difference in the element spacing that det ermines the RNA polymerase specificity of the gene. In this study we h ave analyzed the effect of spacing mutations on activity of Arabidopsi s U2 and U6 genes in transfected protoplasts of Nicotiana plumbaginifo lia and in stably transformed tobacco. In the pol III- transcribed U6 gene the insertions and deletions of either odd or even numbers of hal f helical turns completely inactivate transcription in transfected pro toplasts, consistent with the high conservation of the element spacing found in all plant U-snRNA genes. Surprisingly, while insertions of 5 0 base pairs (bp) or more into the spacer of the pol II-specific U2 ge ne inactivate transcription, a deletion of 5 bp or insertions of as mu ch as 20 bp decrease transcription by only 40 to 70%. This relaxed req uirement for the conserved element spacing is only seen in transfected protoplasts since the same mutant U2 genes are not transcribed in sta bly transformed tobacco when transcription takes place from the chromo some. The results provide some clues about possible factor interaction s at the promoters of plant U-snRNA genes and also offer an example of major differences in transcription between transiently and stably tra nsformed cells.