IDENTIFICATION OF MULTIPLE PROMOTERS WITHIN THE N-RAS PROTOONCOGENE

N-ras possesses a 'housekeeping' promoter, being G + C-rich and devoid of a TATA-box. Transcription initiates at a number of locations withi n this gene, a phenomena that is generally attributed to the absence o f a TATA-box. In this report we investigate the possibility that multi ple promoters, which could potentially contribute to the observed 5' e nd heterogeneity, exist within the murine N-ras gene. The 5' region of the gene was subdivided into several fragments, each corresponding to a region in which one or more transcription initiation site(s) had be en mapped, and the ability of each fragment to express a reporter gene was assessed. Promoter activity was found associated with three indep endent, non-overlapping fragments, two of which were located entirely within transcribed regions of the gene. We found that these intragenic promoters were able to express the N-ras gene itself, as well as the reporter gene. In addition, we found that the activity of an intrageni c promoter fragment was dependent upon the presence of regions encompa ssing initiation sites, and that a small fragment (similar to 40 bp) e ncompassing several initiation sites possessed promoter activity. Thes e data support the existence of an 'initiator' element within the N-ra s gene. Overall, our results demonstrate that multiple promoters resid e within N-ras and suggest that they may play a role in generating the observed mRNA 5' end heterogeneity. The identification of multiple pr omoters within N-ras may have important implications regarding the reg ulation of expression of this gene in normal and malignant tissues. In addition, since a number of other genes with housekeeping promoters a lso initiate transcription at multiple locations, it is possible that the utilization of multiple promoters may represent a common feature o f this class of genes.