Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

Abstract

The discovery of constituents of eukaryotic transcription systems was enabled by the development of in vivo and in vitro methods for their study. Depending on the organism to be studied, the relative importance of each of these two general approaches has been different. Our knowledge about transcription of human genes would be considerably smaller without the results that were obtained by employing in vitro methods. Here, we will mainly focus on in vitro studies of gene expression that contributed to discoveries in the human RNA polymerase (RNAP) III transcription system.In contrast to unicellular yeast, it has until today been and will most likely remain for a many more years impossible to study human RNAP III transcription in cells that have been grown under conditions resembling at least approximately to an in vivo situation in a human being. For that reason, it has hitherto been difficult to address questions concerning the physiological regulation of human RNAP III transcription in its natural environment. However, the identification of the basal components of the RNAP III transcription system could be achieved without directly resorting to a human "in vivo model system." Yet, the identification of human RNAP III subunits and of accessory transcription factors repeatedly took advantage of in vivo systems that were established in other species. Many discoveries of components of the human RNAP III transcription system were fostered by work performed in unicellular (for example, S. cerevisiae and S. pombe) and multicellular eukaryotes (among others, X. laevis or D. melanogaster). Often, results obtained in these model systems helped researchers identify proteins of the human RNAP III transcription system. For instance, the amino acid sequences of transcription factors that were identified and cloned by employing in vivo and in vitro methods in these organisms served as guide for comparing and identifying orthologous transcription factors in human cells (e.g., TFIIIA, TFIIIB [BDP1], TFIIIC35 [GTF3C6]). However, several of the human RNAP III transcription factors were not cloned by homology, but due to extensive purification from human cells (TFIIIB [BRF2], PTF/SNAPc, TFIIIC [GTF3C1-5]). Purification of these factors by employing biochemical methods was only possible, because functional assays were developed that allowed detecting their specific activities. These assays included in vitro transcription, electrophoretic mobility shift assays (EMSA) and footprinting techniques (descriptions of these techniques are found in 1-3 ). Two techniques were essential for the biochemical purification and functional analysis of human transcription factors: (i) the elaboration of protocols that allowed deriving protein extracts from human cells and (ii) the development of cell-free in vitro transcription assays. [4][5][6] Template DNA for in vitro transcription was provided by genes cloned into plasmid DNA. In the case of RNAP III transcription, usually complete and generally short transcribed sequences were included into the...

Leukodystrophy-associated POLR3A mutations down-regulate the RNA polymerase III transcript and important regulatory RNA BC200

“…However, contrary to what we observed in HEK293 cells, 7SL RNA levels were not decreased in mutant MO3.13 cells (Fig. S9), suggesting cell type–specific effects that could be related to upstream regulatory sequences specific to 7SL genes (31). In sum, we obtained two mutant cell lines with decreased levels of BC200 RNA (∼50% reduction in POLR3A M852V and absent in BC200 KO ), which was confirmed by Northern blotting (Fig.…”

“…5 a and Table S4). In contrast, vault RNA levels were comparable across both groups, making them the only transcripts with type 2 internal elements that were not decreased in mutants, which could be due to their vault RNA-specific distal regulatory sequences (31). Consistent with a selective effect on type 2 transcripts, none of the genes coding for type 1 (5S rRNA) or type 3 transcripts (U6 RNAs, Y RNAs, RPPH1, RMRP, and RN7SK) displayed significant differences between control and mutant samples, although some showed a small trend toward decreased expression in mutants (Fig.…”

“…We next examined the other transcripts with type 2 internal promoter elements, which include BC200 RNA, 7SL RNAs, and vault RNAs (28, 29, 31). BC200 (also called BCYRN1 ) and genes encoding 7SL RNA were among the most significantly down-regulated genes in the large RNA transcriptome (Fig.…”

“…The type 1 promoter, which is exclusive to 5S rRNA genes, and type 2 promoters (tRNAs and a few others) contain internal elements located within the transcribed sequence (29). A handful of genes that possess type 2 promoters (BC200, 7SL, and vault RNAs) also have upstream promoter elements (30, 31). In contrast, type 3 genes ( U6, RN7SK, RPPH1, and RMRP ) exclusively possess upstream promoter elements (29, 32).…”

RNA Polymerase III Accurately Initiates Transcription from RNA Polymerase II Promoters in Vitro

“…The TATA Box or an AT-rich Sequence Is Sufficient to Recruit Human Polymerase II and III to Polymerase II Promoters-DNA sequence analysis of the Pol II promoters used in this study revealed the absence of typical Pol III promoter elements Box A/B/C, and PSE (4,45). It was notable, however, that the Pol II promoters from which Pol III initiated contained either a TATA box or an AT-rich sequence around bp Ϫ30 relative to the transcription start site.…”