Identification of multiple promoters within the N- ras proto-oncogene

Abstract

N- ras possesses a ‘housekeeping’ promoter, being G + C-rich and devoid of a TATA-box. Transcription initiates at a number of locations within this gene, a phenomena that is generally attributed to the absence of a TATA-box. In this report we investigate the possibility that multiple promoters, which could potentially contribute to the observed 5′ end 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 been mapped, and the ability of each fragment to express a reporter gene was assessed. Promoter activity was found associated with three independent, 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 intragenic promoter fragment was dependent upon the presence of regions encompassing initiation sites, and that a small fragment (∼ 40 bp) encompassing several initiation sites possessed promoter activity. These data support the existence of an ‘initiator’ element within the N- ras gene. Overall, our results demonstrate that multiple promoters reside within N- ras and suggest that they may play a role in generating the observed mRNA 5′ end heterogeneity. The identification of multiple promoters within N- ras may have important implications regarding the regulation of expression of this gene in normal and malignant tissues. In addition, since a number of other genes with housekeeping promoters also initiate transcription at multiple locations, it is possible that the utilization of multiple promoters may represent a common feature of this class of genes.