DOMAINS OF THE RAT RDNA PROMOTER MUST BE ALIGNED STEREOSPECIFICALLY

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G --> A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant - 7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95. The second site that demonstrated a cyclical response to distance-altering mutations lies between -60 and -55, i.e., between the 3' border of the UPE and the 5' boundary of the CPE. Insertions or deletions of half-multiples of the helical repeat within these sites inactivated the promoter, whereas the insertion or deletion of 10 or 11 bp was permitted. These observations indicate that there is a required stereospecific alignment between the UPE and the CPE and possibly within the UPE.