Studies of the determination of RNA polymerase specificity on a human U6 snRNA gene promoter

Abstract

The proximal promoter of a human U6 small nuclear RNA gene consists of a TATA box and a snRNA proximal sequence element (PSE), and the combination of these two elements directs RNA polymerase III (pol III) transcription. It has been shown in this dissertation that TATA-binding protein (TBP) binds to the U6 TATA box and stimulates U6 transcription. It is conceivable that the TATAbound TBP on the U6 promoter could recruit the rest of the pol 11 general transcription factors such that mRNA-type pol II transcription could occur from the U6 gene. Indeed, a pol II type TBP-TFIIB complex was assembled on the U6 promoter, suggesting that a pol II preinitiation complex can be assembled on the U6 promoter. Furthermore, pol II transcription was detected from the U6 gene when a nuclear extract was employed for in vitro transcription. The pol 11-specific transcription was independent of the PSE, and dependent on the presence of the TATA box. Both pol III- and pol IIspecific transcription were stimulated by addition of recombinant TBP. PSE-binding protein stimulated pol III transcription, but not pol II transcription. Factors that are responsible for differentiating the pathway of pol III and pol II transcription from the U6 promoter were investigated. Octamer element, an upstream activator element of the U6 gene, selectively stimulated pol III transcription over pol II transcription. Furthermore, the effect of Dr2 (human topoisomerase I), a known pol II promoter repressor, was investigated on U6 gene transcription. Dr2 repressed U6 transcription in a S100 extract. However, the repression was overcome by either the presence of an octamer element in the template or by addition of PSE-binding protein (PBP). When Dr2 was tested using a nuclear extract, pol II and pol III transcription were repressed to similar levels. However, pol III transcription was derepressed by the addition of PBP, while pol II transcription remained repressed. These results provide insight into how polymerase specificity might be determined on the U6 promoter.