The Spm transposable element has an unusual, enhancer-insensitive
--Ramesh Raina and Nina Fedoroff
The transpositional activity of the maize Suppressor-mutator (Spm) element is regulated by both positive and negative mechanisms that include alternative splicing of the element's primary transcript, inactivation by an epigenetic mechanism, reactivation mediated by the element-encoded TnpA, and transcriptional inhibition by the same gene product (Fedoroff et al., MNL66:14-15, 1992). TnpA is a DNA-binding protein and there are multiple copies of its 12bp binding site located at the element's 5' end, just upstream from the transcription start site, as well as at the element's 3' end, adjacent to its 13bp terminal inverted repeat sequence. To gain further insight into molecular mechanisms regulating Spm transposition at the transcriptional level, we have identified and analysed the element's promoter sequence using transcriptional fusions to a firefly luciferase gene. Plasmids containing the transcriptional fusions, together with a standard CaMV 35S-chloramphenicol acetyl transferase gene (used to normalize the values obtained with different constructs), were introduced into cultured tobacco cells by microprojectile bombardment (Cook and Fedoroff, MNL66:11-12, 1992). We report here that the Spm promoter has unusual properties that contribute not only to minimizing expression, but which render it resistant to the influence of a nearby enhancer.
Identical levels of promoter activity were obtained with 5' terminal Spm fragments comprising the first 220 or the first 578bp of the element's sequence. These fragments were selected to contain the sequence upstream of the transcription start site at nucleotide 209 (a region we previously designated the 'upstream control region' or UCR), or the upstream sequence together with most of the untranslated GC-rich leader sequence of the first exon (a region we previously designated the 'downstream control region' or DCR) (Banks et al., Genes Dev. 2:1364-1380, 1988). This observation implies that the element's promoter sequence is contained entirely within the sequence upstream of the transcription start site. Progressive deletions through the UCR resulted in the gradual loss of promoter activity. The complete Spm promoter is a weak one, supporting luciferase expression at 1-2% of that observed with the CaMV 35S promoter.
The most surprising property of the Spm element's promoter is that it is insensitive to the influence of a nearby strong enhancer. When the 35S promoter's enhancer was placed upstream of either the Spm promoter consisting of just the UCR or of both UCR and DCR, luciferase activity increased only by a factor of 3-4, in sharp contrast to the 100-200-fold enhancement observed with the 35S minimal promoter. The Spm promoter was converted to a much more highly enhancer-responsive form by introducing an optimal TATA box sequence and by removing the DCR. Although each change had an effect individually, the effects of the two together were more than additive. The sequence that was converted to a TATA box was the TATGAAT sequence at -24 to -30 upstream of the transcription start site identified by others as the element's putative TATA box (Pereira et al., EMBO J. 5:835-841, 1986). This is not a sequence essential for Spm promoter activity, since changes by site directed mutagenesis that either decreased or increased its resemblance to a conventional TATA box sequence had no effect on promoter activity. Nonetheless, even a single base change of the G at -27 to an A residue increased the responsiveness of the Spm promoter to the enhancer, and conversion of the sequence to TATATAA, the 35S promoter's TATA sequence, made the UCR highly responsive to the enhancer. The GC-rich DCR makes a major contribution to the enhancer-insensitivity of the Spm promoter, despite the absence of any perceptible effect of this sequence on the basal activity of its own promoter in the transient assay system.
Thus the inherent properties of the Spm element's unusual promoter region contribute to minimizing transposition frequency and making the element's activity position-independent. That is, Spm's promoter is both a weak TATA-less promoter and it is quite insensitive to enhancement. Taken together with our recent observations that the UCR and DCR are the sites of de novo methylation in transgenic tobacco (see Schläppi and Fedoroff, this issue) and are associated with transcriptional inactivation of the element, these observations suggest that the element's control sequences are indeed primarily or entirely within the 5' terminal 0.6kb UCR + DCR region.
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