A key step in retrieving the information stored in the complex genomes of eukaryotes involves the identification of transcription units and, more specifically, the recognition of promoter sequences by RNA polymerase. In eukaryotes, the task of recognizing nuclear gene promoters and then transcribing the genes is divided among three highly related enzymes, RNA polymerases I, II, and III. Each of these RNA polymerases is dedicated to the transcription of specific sets of genes, and each depends on accessory factors, the so-called transcription factors, to recognize its cognate promoter sequences.RNA polymerase I is unique among the nuclear RNA polymerases in transcribing only one set of genes, the large, tandemly repeated, ribosomal RNA genes, and thus in having to recognize a single promoter structure. RNA polymerase II transcribes the protein-coding genes (mRNA genes) as well as some small nuclear RNA (snRNA) genes. The RNA polymerase II promoters can be divided into a core region, defined as the minimal region capable of directing transcription in vitro, and a regulatory region. The regulatory regions are highly varied in structure, reflecting the highly varied synthesis patterns of cellular proteins and the need for exquisite and complex regulation of these patterns. The core promoters themselves come in different types that, in mRNA-encoding genes, can contain a TATA box, an initiator, a downstream promoter element, or various combinations thereof. The assembly of a functional RNA polymerase II transcription complex on a promoter consisting of just a TATA box has been extensively studied. All the factors involved in the process have been identified, and much is known about how these factors interact with DNA and with each other to recruit, eventually, RNA polymerase II (for reviews, see Orphanides et al. 1996;Woychik and Hampsey 2002). How RNA polymerase II transcription complexes assemble on TATA-less promoters is, however, not as well understood.RNA polymerase III is dedicated to the transcription of an eclectic collection of genes whose main common features are that they encode structural or catalytic RNAs and that they are, as a rule, shorter than 400 base pairs (bp). This length limit is consistent with the elongation properties of RNA polymerase III, which recognizes a simple run of T residues as a termination signal. The genes transcribed by RNA polymerase III encode RNA molecules involved in fundamental metabolic processes, specifically components of the protein synthesis apparatus and components of the splicing and tRNA processing apparatus, as well as RNAs of unknown function. The RNA polymerase III promoters are more varied in structure than the uniform RNA polymerase I promoters, and yet not as diverse as the RNA polymerase II promoters. They have been divided into three main types, two of which are gene-internal and generally TATA-less, and one of which is gene-external and contains a TATA box. Remarkably, we have a good, and in some cases a very detailed, understanding of how RNA polymerase III ...

. Saccharomyces cerevisiae TFIIIB is a complex of three subunits, TBP, the TFIIB-related factor BRF, and the more loosely associated polypeptide ␤؆. Although human homologs for two of the TFIIIB subunits, the TATA box-binding protein TBP and the TFIIB-related factor BRF, have been characterized, a human homolog of yeast B؆ has not been described. Moreover, human BRF, unlike yeast BRF, is not universally required for RNA polymerase III transcription. In particular, it is not involved in transcription from the small nuclear RNA (snRNA)-type, TATA-containing, RNA polymerase III promoters. Here, we characterize two novel activities, a human homolog of yeast B؆, which is required for transcription of both TATA-less and snRNA-type RNA polymerase III promoters, and a factor equally related to human BRF and TFIIB, designated BRFU, which is specifically required for transcription of snRNA-type RNA polymerase III promoters. Together, these results contribute to the definition of the basal RNA polymerase III transcription machinery and show that two types of TFIIIB activities, with specificities for different classes of RNA polymerase III promoters, have evolved in human cells.

Telomerase, an enzyme that maintains telomere length, plays major roles in cellular immortalization and cancer progression. We found that an exogenous BRCA1 gene strongly inhibited telomerase enzymatic activity in human prostate and breast cancer cell lines and caused telomere shortening in cell lines expressing wild-type BRCA1 (wtBRCA1) but not a tumor-associated mutant BRCA1 (T300G). wtBRCA1 inhibited the expression of the catalytic subunit (telomerase reverse transcriptase [TERT]) but had no effect on the expression of a subset of other components of the telomerase holoenzyme or on the expression of c-Myc, a transcriptional activator of TERT. However, endogenous BRCA1 associated and partially colocalized with c-Myc; exogenous wtBRCA1 strongly suppressed TERT promoter activity in various cell lines. The TERT inhibition was due, in part, to suppression of c-Myc E-box-mediated transcriptional activity. Suppression of TERT promoter and c-Myc activity required the amino terminus of BRCA1 but not the carboxyl terminus. Finally, endogenous BRCA1 and c-Myc were detected on transfected mouse and human TERT promoter segments in vivo. We postulate that inhibition of telomerase may contribute to the BRCA1 tumor suppressor activity.

RNA polymerase III (RNA pol III) transcribes many of the small structural RNA molecules involved in processing and translation, thereby regulating the growth rate of a cell. Initiation of pol III transcription requires the evolutionarily conserved pol III initiation factor TFIIIB. TFIIIB is the molecular target of regulation by tumor suppressors, including p53, RB and the RB-related pocket proteins. However, our understanding of negative regulation of human TFIIIB-mediated transcription by other proteins is limited. In this study we characterize a RNA pol III luciferase assay and further demonstrate in vivo that a human homolog of yeast Maf1 represses RNA pol III transcription. Additionally, we show that Maf1 repression of RNA pol III transcription occurs via TFIIIB, specifically through the TFIIB family members Brf1 and Brf2.