UC San Diego

The goal of this study is to better understand the structure-function relationships of the small nuclear RNA activating protein complex (SNAPc), and how this complex is involved in transcription activation and RNA polymerase specificity of small nuclear RNA (snRNA) genes. The SNAP complex is the major component uniquely required for transcription of snRNA genes, some of which are transcribed by RNA polymerase II (Pol II) and some by RNA polymerase III (Pol III). In the fruit fly, SNAPc contains three distinct subunits (DmSNAP190, DmSNAP50, and DmSNAP43) that form a complex prior to binding to DNA; moreover, all three subunits are required for the sequence -specific DNA binding activity of DmSNAPc and each makes direct contact with DNA. Chapter 1 describes truncational analysis that mapped domains within each subunit of DmSNAPc that are involved in complex formation and DNA binding. Our results indicated that the most evolutionarily conserved regions of the subunits were involved in complex assembly. However, domains outside the conserved regions were also important for the DNA binding activity of DmSNAPc, even though they were not required for subunit assembly. Chapter 2 summarizes our present understanding of how snRNA transcription is regulated in the fruit fly, and further compares this knowledge with information obtained from other systems. The structure of snRNA promoters and the contribution of these promoter sequences to RNA polymerase selection were reviewed followed by a discussion of structure-function features of DmSNAPc in comparison to the homologous proteins from other organisms. Evidence that snRNA promoter sequences act as differential allosteric effectors of DmSNAPc conformation was discussed, and how these conformational differences of the DmSNAPc-DNA complex may lead to distinct RNA polymerase specificities of Pol II and Pol III snRNA genes were proposed. Chapter 3 describes studies that investigated the contribution made to DmSNAPc DNA- binding activity by amino acid residues within a novel DNA -binding domain of DmSNAP43. My results revealed that some of the most evolutionarily conserved residues within this domain were essential for DNA binding, whereas other residues made little or no contribution to the DNA binding activity of DmSNAPc