Functional dissection of a mouse ribosomal protein promoter: significance of the polypyrimidine initiator and an element in the TATA-box region.

Abstract

All of the mammalian ribosomal protein (rp) genes examined to date initiate transcription with high precision despite the fact that they do not contain a well-defined TATA box. The initiation sites are situated within polypyrimidine tracts that are flanked by both upstream and intragenic promoter elements. In the TATA-box region of each rp promoter, there is a functionally critical element with nuclear factor binding specificity that is distinct from that of a conventional TATA box. To understand how the various elements contribute to rp promoter function, we have used site-specific mutagenesis-transfection protocols and factor binding analyses to evaluate the significance of the polypyrimidine initiator and the TATA-box counterpart for efficient and accurate transcription of the rpS16 gene. Our results indicate (i) that the polypyrimidine initiator sequence critically defines the position of the transcriptional start site, whereas a much less specific sequence is sufficient to satisfy the efficiency requirement; (ii) that an uninterrupted stretch of pyrimidines in the initiator region is not necessary for efficient transcription of rpS16 gene; and (iii) that the TATA-box counterpart or even a substituted conventional TATA box primarily influences promoter efficiency. The great diversity of promoter design, which is becoming evident as more RNA polymerase II promoters are being carefully dissected, suggests that the requirements for building a functional initiation complex may be much more flexible than was previously appreciated.