Investigation of the RNA polymerase III (pol III) transcription system of eukaryotes was continued, focusing on pol III initiation factors as well as the human La antigen. Pol III produces small RNAs, including the 5S ribosomal RNA and transfer RNAs essential for protein synthesis, as well as certain virus-encoded transcripts. Pol III is a multisubunit enzyme that is directed to its target genes by transcription initiation factors (TF). The human La antigen is a regulatory phosphoprotein that has been shown to serve as a transcription termination factor for pol III, stimulating transcription and directing the maturation of the transcripts, the latter of which is a complex operation that includes end-processing, intron removal, base modification, and proper folding. La ineracts with these transcripts by recognizing their terminal UUU motifs, which result from pol III termination, found at the ends of all newly synthesized pol III transcripts. Patients suffering from systemic lupus erythematosous (a.k.a., Lupus), neonatal lupus, and Sjogrens syndrome produce autoimmune antibodies to the La antigen, although how and why La is targeted for autoantibody production is unknown. Some viral-encoded factors, including the adenovirus E1A protein, modulate the activity of specific pol III TFs, and other virus factors modulate the nucleo-cytoplasmic distribution and activity of La. Understanding the mechanisms by which La and other pol III-related factors function in the pathways used for small RNA production, and how these pathways are controlled during normal cellular proliferation, are major goals of this Section. Experimental systems representing only two organisms are currently available for the study of pol III transcription, the budding yeast, S. cerevisiae, and human. While these share extensive homology in the factors and mechanisms involved in transcription initiation, notable differences exist in the TFs that map to the termination ends of the transcription complex as well as in the minimal termination signals required for pol III termination. Therefore, another goal of this Section is to investigate pol III transcription in the fission yeast, S. pombe, a genetically-tractable organism in which our preliminary studies suggested a pol III termination signal mechanism more similar to that of humans than is S. cerevisiae.Summary of Major Findings:1. A pol III termination- dependent gene was developed for use in S. pombe. This gene encodes an opal suppressor tRNA that can suppress a nonsense codon in the mRNA encoding a purine-synthetic enzyme whose activity is monitored in vivo by a colorimetric plate assay. This reporter gene was shown to require the S. pombe La protein for efficient expression. However, when the 3? end of the precursor tRNA is generated by posttranscriptional processing rather than pol III termination, La is not required for expression. This provides the first evidence that La activity is linked to pol III termination in vivo, and establishes a genetically-tractable system to study this process.2. The gene encoding a putative TFIIIC subunit that is believed to map to the termination end of the transcription complex, was identified and shown to be essential for viability in S. pombe. Immunoaffinity purification of a tagged version of this protein was isolated from S. pombe and shown to have tRNA promoter-specific DNA binding in vitro. 3. The gene encoding a putative pol III-specific subunit of pol III was identified and epitope tagged in S. pombe. Immunoaffinity purification of this subunit yielded a pol III enzyme with activity in vitro. Biochemical and enzymatic properties of this polymerase, including its ability to recognize a minimal pol III terminator element were characterized. 4. Monospecific antibodies directed to a 20-amino acid peptide containing phosphoserine 366 within the human La antigen were developed and characterized by several methods. These antibodies are highly specific in their ability to distinguish the unphosphorylated form of the La antigen from the phosphorylated form both as purified proteins and in the context of wholenuclear extract. 5. Structure-function analysis had previously revealed a novel mode of RNA binding for the human La antigen in vitro that involves recognition of the initiating triphosphate nucleotide of a nascent pol III transcript, by the C- terminus basic region of La.In vivo results now support this model and reveal important distinctions between the La proteins of yeast and human s.6. Several subunits of S. pombe pol III TFs were produced in recombinant bacteria and purified. These subunits will be used to attempt to establish a minimal reconstituted system of S. pombe pol III transcription in vitro.
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