The Section on Molecular and Cell biology studies transcription by RNA polymerase III (Pol III) as a model system of eukaryotic gene expression. Our interests are focused most intensely on individual steps in the pathways of RNA production but also include the mechanisms by which sequential steps are coordinated during RNA biogenesis. Thus, we examine transcription and early posttranscriptional processing events that play a role in active maturation of nascent RNAs before they are exported to the cytoplasm where they function. The Pol III transcripts studied most intensely are the transfer RNAs, the adapters used for translation of the genetic code. We use modern genetics, molecular biology, and biochemical approaches, relying heavily on analytical biochemistry as well as on tissue culture, yeast, and transgenic mice.

Functions of the Human La Antigen in RNA Expression
Intine, Mukhergee, Nelson, Maraia
The human La antigen is a nuclear phosphoprotein that contributes to the production of pol III transcripts. La is present in all eukaryotic cells so far examined. It is a target "antigen" of autoantibodies in patients suffering from systemic lupus erythematosous, neonatal lupus, and Sjögren's syndrome, although how and why La is targeted for autoantibody production remains unknown. In normal cells, La is a component of a Pol III holoenzyme, which participates in transcription termination and remains associated with the newly synthesized transcripts to direct their maturation. In this way, La chaperones nascent RNAs through complex pathways and, in some cases, controls their accessibility to the processing enzymes. La performs its function by high-affinity sequence-specific binding to the nascent RNA 3' UUU-OH motif that is common to all transcripts synthesized by Pol III and results from transcription termination.

Our section mapped the phosphorylation site of La to serine 366 and showed that the phosphorylation interferes with La's ability to interact with the initiating pppG of the nascent transcript and to activate transcription initiation by Pol III, suggesting that such activities are mechanistically related and coordinated in an "La cycle" of transcription and posttranscriptional regulation. Others have recently shown that La is dephosphorylated at S366 early during apoptosis. We have developed monospecific antibodies directed against short peptides containing phosphoserine 366 and have used them to show that unphosphorylated and phosphorylated La have different subcellular and subnuclear localizations. Indeed, we have identified several motifs of La that control nuclear, nucleolar, and cytoplasmic localization. For some motifs, disruption of proper localization causes processing defects that are detrimental to tRNA expression. In summary, our section uses a multitude of approaches to explore the mechanisms that control the function of La (as well as of other) protein(s) to develop an integrated view of eukaryotic gene expression while advancing our understanding of this important protein antigen.

Transcription Termination by RNA Polymerase III
Zhang, Maraia
We have developed a Pol III transcription system in the fission yeast, S. pombe, and have developed and characterized a pol III-dependent tRNA reporter gene. The gene encodes an opal suppressor tRNA that suppresses a nonsense codon in the mRNA encoding a purine-synthetic enzyme (Ade6-704), whose activity can be monitored by an in vivo colorimetric plate assay. We demonstrated that the expression of the gene in S. pombe depends on accurate and efficient termination by pol III and established that the minimal number of dT residues required for efficient termination is five. We then showed that this tRNA gene requires the La protein for efficient expression in vivo. Many important questions remain to be answered, such as the nature of the mechanistic link between La and pol III termination, whether the lack of reporter gene-derived transcripts in the La-minus strain is attributable to a defect in transcription rate, nascent RNA processing, or both, and what other factors contribute to the La-dependent activation of this tRNA gene.

Transcription Initiation by RNA Polymerase III
Huang, McGillicuddy, Maraia
Although a long-standing interest of the section has been transcriptional termination, our recent discoveries and developments have led us to also examine mechanisms of Pol III initiation. Specifically, ongoing studies focus on the two central transcription factors (TF), TATA-binding protein (TBP) and the TFIIB-related factor, Brf, both of which function at the core promoter. Homologs of these factors exist in all eukaryotes and archea and are central in controlling transcription initiation. The core promoter is itself of central importance in transcription because, in addition to directing transcription initiation, core promoters integrate complex input from distal regulatory elements. Genome-wide analysis has revealed that, in contrast to the core promoters of human and S. cerevisiae tRNA genes, which have long been known to be TATA-less, the core promoters of tRNA and 5S rRNA genes in S. pombe contain TATA elements. We used tRNA-dependent suppression and other in vivo assays as well as in vitro transcription to demonstrate an obligatory requirement for upstream TATA elements for tRNA and 5S rRNA expression in S. pombe. We extended our studies to large rRNA synthesis, as mutation of the TATA element in the Pol I promoter also abolished rRNA expression in fission yeast. The results point to TATA-unified transcription systems in contemporary eukaryotes and provide insight into the residual need for TBP by all three Pols in other eukaryotes despite a lack of TATA elements in their promoters. We are using this yeast system to advance further our understanding of basic mechanisms of transcription initiation.