Protein-nucleic acid interactions that guide transcription by eukaryotic RNA polymerases (Pol's) will be investigated in the yeast Saccharomyces cerevisiae. We will focus on the synthesis of small RNAs by Pol III and Pol II. Interaction of the Pol III initiation factors TFIIIB and TFIIIC with conserved promoter elements, and the role of chromatin structure in this process, will be examined in the context of the yeast U6 snRNA gene, SNR6. The involvement of a novel RNA-binding protein, Nrd1, in termination of Pol II transcription on small nuclear and small nucleolar RNA genes will also be investigated. TFIIIB binds upstream of the transcription start site and recruits Pol III. Normally, TFIIIB binds DNA with little sequence-specificity; its binding site is determined by protein-protein contacts with TFIIIC. However, the SNR6 upstream region has the unique ability to specifically position TFIIIB via DNA-protein contacts. We will use SNR6 to study the TFIIIB-DNA interaction in vivo. TFIIIC is an assembly factor for TFIIIB. It binds to the conserved A and B block promoter elements, which are normally located in the RNA-coding region of Pol III-transcribed genes. SNR6 has a unique extragenic B block, which therefore functions only as a promoter element and not as part of an RNA. We will exploit this feature of SNR6 to perform a genetic analysis of the TFIIIC-B block interaction. The SNR6 A and B blocks are separated by 200 base pairs, more than twice as far apart than in any other known gene. We will test the hypothesis that chromatin structure brings the SNR6 A and B blocks closer together to facilitate binding of TFIIIC. Few positively-acting chromatin structures are known and they are unprecedented for Pol III transcription units, so if such a structure is confirmed it will be thoroughly studied. We discovered the essential Nrd1 protein by virtue of its ability to induce 3'-end formation in a nascent Pol II transcript upon recognition of a specific RNA sequence. We will test the hypothesis that Nrd1 associates with elongating Pol II and directs the termination of synthesis of non-polyadenylated Pol II transcripts, such as the small nuclear and small nucleolar RNAs. A human homolog of Nrd1 will be sought. These studies will provide fundamental insights into the mechanism of transcription initiation and termination, which are key steps in the control of eukaryotic gene expression.
