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

Abstract

Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription.

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.^4-6 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 template DNA. The genes transcribed by RNAP III and the promoters recognized by its cognate transcription factors have been reviewed^7,8 and are schematically depicted in Figures 1–3. From cellular extracts, individual transcription activities were purified and used for reconstitution of RNAP III transcription in vitro. In 1980, Segall and colleagues described the reconstitution of human tRNA and 5S RNA transcription in vitro by two (tRNA) or three (5S RNA) partially purified protein fractions, respectively. According to their elution profile from phosphocellulose (P11 or PC) chromatography, these fractions were referred to as P11 (or PC) fractions -0.1 (-A), -0.35 (-B) and -0.6 (-C), containing the transcription activities TFIIIA (P11–0.1), TFIIIB (P11–0.35) and TFIIIC (P11–0.6). RNAP III eluted in fractions P11–0.35 and P11–0.6.⁹ From these fractions, the individual transcription activities were further purified over several subsequent chromatographical steps. In the following, we will give a short and non-exhaustive overview over some key-steps in the identification and cloning of human RNAP III transcription factors that have been made possible by biochemical purification and assay of their activities by in vitro transcription.

Figure 1. Schematic representation of the regulatory elements of human RNA polymerase III transcribed type 1 and type 2 genes. Nucleic acid nomenclature: R = G/A; Y = C/T; W = A/T. Designations of individual boxes are represented in appropriate color codes below type 1 and 2 gene schemes. Type 1 gene: 16 out of 17 human RNA5S genes retrieved at www.ncbi.nlm.nih.gov/nucleotide are identical; T84 within the C-box of RNA5S9 is substituted by a C. The sequence blocks of A- and C-boxes, as well as of the intermediate element (IE) in the human 5S gene are chosen by analogy to described sequence blocks in the X. laevis 5S RNA gene.^129,130 The 5S gene A-box sequence lacks the conserved T in the first position of the tRNA consensus A box. The human D-box element sequence upstream of the transcription start site of the 5S gene was published.¹³¹ Type 2 genes: are representatively depicted by human tRNA consensus sequences that have been derived from published data.^132-134 tRNA termination occurs at variable sites downstream of the B-box.

Figure 3. Schematic representation of human RNA polymerase III transcribed genes with regulatory elements 5′ and 3′ of the transcription start site (type 4 genes). Nucleic acid nomenclature: K = G/T; Y = C/T; R = G/A; S = G/C; W = A/T. Nucleotide sequences of individual presumably regulatory elements are shown for each gene. Designations of individual boxes are represented in appropriate color codes below type 4 gene schemes. Experimental proof for the functionality of these elements has been shown for the ATF/CRE element in the human 7SL and herpesvirus 4 EBER genes.^{89,90,121,138} Inter-species sequence conservation was shown for VTRNA1–1, -2, -3 and VTRNA2–1 regulatory elements. Vault distal elements comprise the shown sequences and two distinct additional further distal sequence blocks for each gene.^139,140 PSE and STAF-binding sites in the human TRNAU1, RN7SL1 and 2, as well as the BCYRN1 gene were delineated by sequence homology with PSE and STAF-binding site consensus sequences (Fig. 2). TATA-like elements were proposed for the VTRNA1–1, -2, -3, the 7SL1 and 2 genes, EBER genes (ETAB)¹³⁸ and the mouse analog of the BCYRN1 gene.^121,122,140 Sequences at similar positions in VTRNA2–1 and TRNAU1 genes are shown. A stimulatory influence of a SP1 binding site on in vitro transcription of the EBER2 gene was shown.¹³⁸ An octamer element as a potential enhancer element was identified by sequence homology upstream of the BCYRN1 gene. Gene internal A and B boxes are appropriately depicted within the transcribed regions. Bases that differ from the tRNA consensus sequences (Fig. 1) for these elements are shown in black letters.

The General Human RNA Polymerase III Transcription Machinery

TFIIIA

The first description of a human transcription activity being exclusively required for the expression of the 5S gene in vitro was published in 1980 by R.G. Roeder and colleagues (Segall et al., 1980). The authors showed that 5S gene transcription requires crude fractions containing transcription factors TFIIIA (PC-A), TFIIIB (PC-B), TFIIIC (PC-C) and RNAP III (PC-B and PC-C), whereas tRNA transcription was reconstituted by the fractions containing TFIIIB, TFIIIC and RNAP III only. Ordered association of TFIIIA, TFIIIC and TFIIIB with the 5S gene was established by in vitro studies using partially purified human factors.^10,11 The identification of the amino acid sequence of human TFIIIA¹² and molecular cloning of the corresponding cDNA^13,14 demonstrated high homology (57% identity) to X.laevis TFIIIA, which had been identified and cloned earlier.^15,16

TFIIIB

Starting with phosphocellulose fraction B (PC-B or P11–0.35), several research groups were involved in the purification of human TFIIIB. K.H. Seifart and colleagues published in 1988 the partial purification of human TFIIIB.¹⁷ After the molecular cloning of the TATA-binding protein (TBP) from human cells^18,19 and the discovery that TBP participates in U6 transcription in yeast²⁰ and human cells^21-23 it became clear that it is also involved in the transcription of RNAP III genes with internal promoter elements.²⁴ Subsequently, TBP and associated factors were purified that reconstituted TFIIIB activity at gene internal promoters.^25-29 Further purification resulted in the identification of human TFIIB-related factor 1 (BRF1/TFIIIB90),^30,31 showing extensive sequence homology in its N-terminal half to the orthologous protein from S. cerevisiae^32-34 and to the paralogous RNAP II transcription factor TFIIB.^35,36 Depletion of TBP from cellular extracts led to the loss of in vitro transcription of all genes expressed by human RNAP III. However, reconstitution of transcription from gene internal promoter elements required a complex of TBP and associated factors (TAFs), whereas transcription of gene regulated by external promoters could be reconstituted by purified recombinant human TBP only.^25,29 These results suggested the existence of distinct TFIIIB activities that are specifically required for either in vitro transcription of gene internal promoter-containing genes or for that of genes with promoter elements in the 5′ non-transcribed region. In 1995, these two activities were physically separated by chromatography.³⁷ The proteins constituting both TFIIIB-activities were identified and cloned.^38-41 TFIIIBα, being active in transcription of genes with promoter elements 5′ of the transcription start site (TSS) is composed of TBP, TFIIIB double prime (BDP1) and BRF2. TFIIIBβ, required for transcription of genes with gene-internal promoter elements, is composed of TBP, BDP1 and BRF1.

TFIIIC

TFIIIC was initially described as primary DNA-binding protein at tRNA and adenoviral VA genes (A- and B-box-containing gene internal promoter elements) and as secondary DNA-binding factor of the 5S gene (A- and C-box-containing gene internal promoter). Later, human TFIIIC-activity was chromatographically separated into two fractions that either comprised the DNA-binding activity (TFIIIC2) or that contained an essential, yet not primarily DNA-binding activity (TFIIIC1).^1,42 The six subunits constituting the DNA-binding complex were identified^43-46 and the corresponding cDNAs were cloned.^47-52 Reconstitution of in vitro transcription by partially purified fractions indicated that TFIIIC1 may be related to the BDP1 subunit of TFIIIB, a suspicion that was corroborated by the fact that a C-terminally truncated version of BDP1 (recombinant human TFIIIB150) was able to partially replace TFIIIC1 in in vitro transcription assays.⁵³

PBP/PTF/SNAPc

As for TFIIIC, also the identification of an activator of U6 transcription was achieved by purification from P11–0.6 (PC-C) fractions and analysis of its activity by in vitro assays, including DNase I footprinting, EMSA and in vitro transcription. This transcription factor, interacting with the proximal sequence element (PSE) of U6 and 7SK gene regulatory sequences was discovered due to its ability to reconstitute transcription of these genes in vitro. This factor, described as PSE-binding protein (PBP),²³ PSE-binding transcription factor (PTF)⁵⁴ or small nuclear RNA-activating protein complex (SNAPc)⁵⁵ binds to the PSE and is required, together with OCT-1 for full activation of U6 and 7SK genes. The subunits of this complex were purified and their corresponding cDNAs were cloned.^56-62 The human RNAP III transcription factors and the cloning of their subunits are listed in Table 1.

Table 1. Listing of the human RNA polymerase III transcription factor subunits and the papers describing the cloning of the respective cDNAs.

Transcription factor	Subunits	Cloning described in ref.
TFIIIA	TFIIIA/GTF3A	13,14
TFIIIB	TBP	18,19
	BRF1 (TFIIIB90)	30,31
	BRF2 (TFIIIB50)	40,41
	BDP1 (TFNR; TFIIIB150)	39,40
TFIIIC	TFIIIC220/GTF3C1	47,48
	TFIIIC110/GTF3C2	49
	TFIIIC102/GTF3C3	50
	TFIIIC90/GTF3C4	51
	TFIIIC63/GTF3C5	50
	TFIIIC35/GTF3C6	52
PBP/PTF/SNAPc	PTFα/SNAPC4	61
	PTFβ/SNAPC3	59,60
	PTFγ/SNAPC1	56,58
	PTFδ/SNAPC2	57,58
	-/SNAPC5	62

RNA polymerase III

Human RNA polymerase III was purified from HeLa cells and the cDNAs encoding its subunits were cloned.^63-66 It was shown that two isoforms of RNAP III are present in human cells that are both capable of transcribing the three distinct types of RNAP III promoters in vitro, but that differed in their cellular expression patterns and functions.^67,68 Specific functions in transcription initiation could be assigned to a ternary subcomplex of RNAP III. From the complete 17 subunit-containing enzyme, three subunits (RPC32; RPC39; RPC62) could be separated either by treatment with 4M urea or by sucrose gradient centrifugation. It was shown by in vitro transcription of intact RNAP III genes or of “tailed templates” that the remaining 14 RNAP III subunits were catalytically active but no longer capable of specifically initiating transcription from RNAP III promoters. These results showed that the ternary subcomplex of human RNAP III fulfills important roles in transcription initiation.⁶⁴

Transcriptional Regulators of Human RNA Polymerase III Transcription

Repressors

Several transcriptional activators, co-activators, but also repressors of human RNAP III transcription in vitro have been identified, indicating that transcription by RNAP III may be fine-tuned according to the needs of a cell. Tumor suppressor proteins (e.g., TP53; RB1; PTEN; CDKN2A (ARF)) have been implicated in the inhibition of RNAP III transcription.^69,70 In addition, negative regulators of human RNAP III transcription with no described tumor suppressor protein function were identified. These negative regulators of RNAP III transcription may, among other functions, adapt transcription levels in response to cellular stresses. For instance, in response to cell growth and nutrient availability, and similar to what was demonstrated in the yeast S. cerevisiae,⁷¹ MAF1 was shown to be a general repressor of human RNAP III transcription.^72-76 Other examples of RNAP III transcriptional inhibitors include ACR1 and YY1, which were shown to inhibit the expression of Alu elements.^77,78 In the case of the ACR1 protein, it was furthermore shown that its inhibitory activity was specific for Alu genes and that it did not repress tRNA or RNAP II-driven transcription.⁷⁷ For the YY1 protein, however, also positive roles in RNAP III transcription have been proposed, such as facilitating SNAPc/PTF-binding to the PSE of the U6 gene⁷⁹ and a possible binding to the A-box of the tRNA^Gln gene.⁸⁰ DR1 (also known as negative cofactor 2 β [NC2beta]) and its dimerization partner DRAP1 (NC2alpha) were shown to associate with and repress RNAP III transcription.^81,82 In addition to these repressors that may act by regulating the binding of RNAP III transcription factors to their cognate DNA elements and/or by affecting their interaction with the components of the RNAP III transcription machinery, there are also reports of repressors that act on the level of DNA methylation or chromatin-related mechanisms. Besides the KRAB-domain containing factor KOX1 that represses RNAP III transcription in cellular transfection assays,⁸³ a repressive function was shown for the DNA methyl transferases DNMT1 and DNMT3a in human U6 transcription in vivo. This repression was recapitulated in transcription of the U6 gene in vitro by methylating template DNA with the heterologous Spiroplasma DNA methyltransferase M.SssI.⁸⁴

Activators

The first example of an enhancer element stimulating RNAP III transcription was the octamer binding site in the U6 and 7SK gene 5′ flanking sequences.^85-87 Shortly after recognizing a role for the octamer element in RNAP III transcription, it was shown that RNAP II transcriptional activator proteins, OCT-1 and OCT-2, bound to this site and stimulated transcription by RNAP III.^54,88 Thereafter a couple of other examples of activator proteins stimulating RNAP III transcription have been published, including ATF (in transcription of Epstein Barr virus EBER1 and 2 genes⁸⁹and of the 7SL gene⁹⁰), SP1 (EBER genes)⁸⁹ and MYC (tRNA^Leu gene; VA1 gene).⁹¹ The identification of the SPH element as an enhancer of the tRNA^Sec gene and of ZNF143 (STAF), the transcriptional activator binding to this gene, was achieved in X. laevis.^92,93 Several years later the human gene encoding ZNF143 was cloned.^94,95 These discoveries demonstrated that several distinct activators could interact with and stimulate RNAP III transcription. However, and probably due to the small number of genes transcribed by RNAP III that are directed by promoters with regulatory elements located 5′ of the transcription start site, also the number of transcriptional activators that have been described remains limited.

Chromatin-associated regulation of RNAP III transcription

Compared with RNAP II transcription, only few in vitro studies of human RNAP III transcription were conducted with template DNA that was reconstituted into chromatin. TFIIIA was shown to prevent the assembly of chromatin on the 5S gene, but that preassembled chromatin excluded subsequent binding by TFIIIA.⁹⁶ TFIIIC was shown to possess intrinsic acetyltransferase activity (GTF3C4/TFIIIC90 and GTF3C3/TFIIIC102) and to alleviate chromatin-mediated repression in the presence of HeLa nuclear extract.^51,97 Thereafter, expression of tRNA and VA1 genes was analyzed by employing an in vitro transcription system that was reconstituted with recombinant and highly purified transcription factors, RNAP III and a chromatin assembly system consisting of purified HeLa histones, ACF and NAP1. In this system, it was shown that the acetyltransferase P300 is recruited to RNAP III genes and activates transcription.⁹⁸ The authors showed that P300 is recruited by TFIIIC, probably involving a direct interaction of P300 with the GTF3C2 (TFIIIC110) subunit. The interaction stabilized TFIIIC-binding to tDNA in an acetyltransferase-independent manner. Surprisingly, and in contrast to what was shown for RNAP II transcription, P300 also stimulated tRNA in vitro transcription from a naked DNA template.⁹⁸ P300 could not be replaced by PCAF in this reconstituted in vitro chromatin transcription assay. In contrast, the PCAF-related GCN5 complex (STAGA/TFTC)⁹⁹ was shown to stimulate transcription of 5S and tRNA genes in vivo.¹⁰⁰ In the case of U6 transcription, it was suggested that a nucleosome positioned between the DSE and the PSE approaches these to elements, thereby activating in vitro transcription of this gene.^101,102 This model was confirmed through analyzes of the 7SK gene by employing in vivo footprinting techniques.¹⁰³ In addition, the U6 gene was shown to be regulated by the chromodomain helicase DNA-binding protein 8 (CHD8). This chromatin remodeling factor was demonstrated to associate with ZNF143 (STAF) and to contribute to efficient in vitro expression of the human U6 gene that was reconstituted into chromatin.¹⁰⁴

Co-activators

Positive co-factor 4 (PC4) was initially shown to stimulate RNAP II transcription in vitro.^105,106 Several years later it was shown that PC4 acts also as co-activator of RNAP III transcription.¹⁰⁷ Comparable to PC4, also topoisomerase 1 could be co-immunoprecipitated with an affinity-purified holo-TFIIIC complex and was shown to act as a positive cofactor of RNAP III transcription. Both PC4 and Topo1 were shown to reinforce interactions of TFIIIC with downstream promoter sequences. In addition, it was demonstrated for PC4 that it stimulates multiple round, but not single round transcription of the VA1 gene in vitro.¹⁰⁷ Analogous to PC4, it was reported that Sub1, the S. cerevisiae ortholog of PC4, associates with RNAP III transcribed genes and enhances their expression.^108,109

Distinction of Components Required for Basal or Activated RNAP III Transcription

RNAP III genes with promoters 5′ of the transcription start site

Due to the close physical association and fixed distance of the PSE with the TATA-box and the transcription start site (Fig. 2) and probably also because of the functional similarity to TFIIIC in driving RNAP III transcription from promoters that are regulated by gene internal promoter sequences, PTF/SNAPc has often been described as general (basal) transcription factor.^{54,56,79,110-112} However, in vitro transcription experiments with 7SK- or U6-promoter deletion mutants showed that accurate, RNAP III-dependent transcription initiation, elongation and termination can be directed by sequences located about 40 nucleotides upstream of the transcription start site only. Thus, basal in vitro transcription of these genes does not require the PSE or DSE, but is exclusively dependent on the TATA-box and possibly surrounding nucleotides.^38,113 Taking these results into account, the TATA-box should be regarded as the only explicitly described promoter element for U6 and 7SK genes (and possibly for all RNAP III-transcribed genes with promoter elements 5′ of the TSS). Accordingly, the PSE and its interacting PTF/SNAPc in turn should be regarded as a pair of enhancer sequence and interacting transcriptional activator complex. In support of a possible transcriptional activating, rather than basal transcription factor-like function of PTF/SNAPc is the finding that at least one subunit of the SNAPc/PTF acts as (co-)activator of mRNA transcription. It was shown that the SNAPC1 (PTFγ), but not the SNACP4 (PTFα) subunit of this complex co-localizes with actively transcribed protein-coding genes in a manner that depends on active RNA polymerase II transcription elongation. Depletion of SNAPC1/PTFγ resulted in reduced levels of activated transcription, but did not affect basal transcription of these genes. Many of the genes depending on SNAPC1/PTFγ for activated transcription were regulated by the epidermal growth factor (EGF) and retinoic acid (RA) signaling pathways.¹¹⁴ These results indicate that at least parts of the SNAPc/PTF complex may act as a transcriptional (co-)activator rather than a basal transcription factor.

Figure 2. Schematic representation of regulatory elements of RNA polymerase III transcribed type 3 genes. The sequences and regulatory elements located 5′ of the transcription start site of the H1 (RPPH1), MRP (RMRP), U6ATAC (RNU6ATAC), U6–1 to U6–9 (RNU6–1, -2, -7, -8 and -9), 7SK (RN7SK), Y1 to Y5 (RNY-1, -3, -4 and -5) are depicted. Consensus sequences of the octamer element (red), STAF-binding site (blue), proximal sequence element (PSE; green) and TATA-like sequences (orange) are shown below. Nucleic acid nomenclature: B = T/C/G; D = A/T/G; H = A/C/T; V = C/A/G; K = G/T; M = A/C; S = G/C; W = A/T; Y = C/T; R = G/A; n = any of the four bases. Typical distances between the transcription start site (TSS) and regulatory boxes are indicated below the representation of the genes. The distances spanned by individual DNA elements, as well as those between the TSS and the TATA-like box and between the TATA-like box and the PSE are indicated by arrows and number of nucleotides (nt). The distal sequence element (DSE) is typically found 175 to 248 nt upstream of the TSS and composed of an octamer element and a STAF binding site. Octamer element and an inverted STAF-binding site are located closer to the transcription start site of the RPPH1 gene. The RMRP gene possesses an octamer element only at around -215 and the RNU6ATAC gene that comprises a potential STAF-binding site around -110. Sequences showing homology to inverted STAF-binding sites are located at -420 and -470, respectively, relative to the transcription start site of the RMRP gene (not shown). STAF (ZNF143) binding to the human U6 and H1 genes was demonstrated.^94,95 The other STAF-binding sites have been deduced by sequence homology. PBP/PTF/SNAPc-binding to human U6 and 7SK genes has been demonstrated.^26,54,55 Potential PSEs in the other gene-regulatory sequences have been deduced by sequence homology. OCT-1 binding and stimulation of in vitro transcription of human 7SK and U6 genes was demonstrated.^54,135 Deletion of the octamer element caused moderate effects on in vitro transcription of the RPPH1 gene.^136,137

RNAP III genes with promoters within the transcribed sequence

It is also an unresolved question which DNA elements represent the basal human RNAP III promoter elements of genes that do not require gene external promoter or enhancer elements. However, results of studies that have been conducted in the yeast S. cerevisiae shed some light on this question. In S. cerevisiae, the U6 gene comprises promoter elements that are located 5′ of the transcription start site (TATA-box), a weak A-box within the transcribed region as well as a B-box downstream of the transcription termination site (reviewed in¹¹⁵). Comparable to promoter recognition in tRNA genes, TFIIIC binds to the B-box downstream of the U6 gene and positions TFIIIB around the transcription start site, permitting the recruitment of RNAP III. Both S.cerevisiae U6 and tRNA genes utilize the A-box for determining the transcription start site in vivo. In the case of the U6 gene, a TATA-box contributes to transcription start site selection in vitro.¹¹⁶ The transcription of S. cerevisiae tRNA genes was also shown to be regulated by T/A enriched sequences upstream of the TSS.¹¹⁷ Thus, besides the fact that the B-box is either located within the transcribed region (tRNA genes) or downstream of the transcription termination site (U6 gene), the overall structure of gene regulatory elements is similar in both cases. Yeast TFIIIC is composed of two well characterized sub-modules, τA and τB, that respectively recognize the A- (τA) or B-box (τB).¹¹⁸ It was shown that the distance of A- and B-boxes can be increased from 74 to 365 nucleotides without affecting transcription efficiency. However, mutations in the A-box were not tolerated if the A- to B-box distance was increased, whereas the wild type promoter architecture was still active with the same A-box mutations.¹¹⁹ As a consequence, TFIIIC may be regarded as a complex that integrates functions of a general transcription factor (GTF; A-box binding; τA) and of a transcriptional activator (B-box binding; τB.)¹¹⁵ Importantly, it was also experimentally shown that the A-box of the SNR52 gene is sufficient for directing S. cerevisiae RNAP III transcription, supporting a functional specialization of submodules within TFIIIC into GTF (τA) and transcriptional activator (τB).¹²⁰ In accordance with this model, the B-Box element within the transcribed regions of genes with type 2 promoter elements may be considered as an enhancer element. Since the subunit composition of yeast and human TFIIIC has been conserved during evolution,⁵² it can be assumed that this point of view may also hold true for human RNAP III transcription although this has not yet formally been shown.

RNAP III genes with promoter sequences within the gene and 5′ of the transcription start site

RNAP III transcribed genes containing not only promoter elements located 5′ of the TSS, but also regulatory elements within the transcribed region have been described. These genes have been identified in humans and other higher eukaryotes and their gene regulatory elements are also sometimes referred to as type 4 promoters (e.g., 7SL RNA [SRP], vault RNA [HVG], BC200 RNA [BCYRN1] and Epstein-Barr virus encoded EBER RNA genes; Figure 3).^{7,8,89,90,121} These genes possess not only canonical A- and B-boxes appropriately positioned within the transcribed region of the gene, but also promoter and enhancer elements, such as the TATA-box, PSE, DSE, ATF- or SP1-binding sites in several distinct individual combinations upstream of the TSS (Fig. 3). Promoter elements upstream and downstream of the transcription start site are of different importance for the transcription of these genes in vitro or in vivo. It was shown that efficient in vitro transcription of the mouse BC1 analog of human BC200 in HeLa nuclear extracts only requires gene internal promoter elements. In contrast, in vitro transcription with rat cortex nuclear extracts was additionally dependent on DNA elements upstream of the TSS.¹²² These results were confirmed by transfection of the prosimian G22 ortholog of human BC200 into HeLa cells¹²³ or by analysis of 5′ regulatory element deletion mutants in transgenic mice.¹²⁴ The first 22 nucleotides upstream of the TSS were indispensable for the expression of the G22 gene, demonstrating that they comprise promoter elements. The efficiency of BC1 transcription, however, was further increased if more than 22 nucleotides upstream of the transcription start site were included into the expression constructs, indicating that these sequences contained enhancer elements.¹²³ In addition, it was demonstrated that sequences more than 250 nucleotides upstream of the TSS were important for brain-specific expression of the BC200 gene.¹²⁴ In line with the importance of gene regulatory elements 5′ of the TSS for genes with type 4 promoters is the observation that the HVG4 (VTRNA3–1) pseudogene lacks such regulatory sequence elements and is not expressed in several cell lines although it does contain functional gene internal type 2 promoter elements.¹²⁵

Mechanistic aspects of activated RNAP III transcription

Fundamentally, the mechanisms of transcription by RNAP III, and in particular the discrimination of basal and activated transcription, seem to be very similar to those that have been described for RNAP II transcription. However, an important difference between RNAP II and RNAP III transcription concerns the distance of enhancer elements to the transcription start site. This distance is very limited in RNAP III transcription and does not exceed several hundreds of base pairs (Figs. Two and 3).⁷ In contrast, in RNAP II transcription enhancer-promoter-distances of several tens of thousands of base pairs have been described and mechanisms including DNA looping have been proposed for bringing enhancer and promoter sequences in close vicinity. DNA looping may require the participation of protein-protein contacts as well as protein-DNA interactions of a multi-subunit transcriptional co-activator complex, the mediator.¹²⁶ Thus, the difference in the localization of enhancer sequences relative to the promoter elements in transcription by RNA polymerases II or III may largely be explainable by the utilization of the mediator complex in RNAP II transcription and simple activator-basal transcription factor-interactions in the RNAP III transcription system. The lack of the participation of the mediator complex in RNAP III transcription may also explain the fact that no enhancer elements have been identified downstream of the transcribed region so far for human RNAP III genes that are regulated by promoters located upstream of the TSS.

Conclusions

In vitro transcription assays have been instrumental for the identification and characterization of the proteins that constitute the human RNA polymerase III transcription machinery. In addition, by employing promoter deletion constructs and partially purified reconstituted in vitro transcription systems, the minimal regulatory sequence requirements for human RNAP III genes with promoters 5′ of the transcription start site could be defined (sequences comprising about 40 nucleotides upstream of the TSS, including the TATA-box). In the case of gene internal promoters, the A-box is the only essential promoter element in S. cerevisiae and due to the high degree of conservation of the proteins required for tRNA transcription in yeast and humans, the situation is probably similar in humans. Therefore, TATA- and A-boxes may be regarded as the only clearly defined RNAP III promoter elements, whereas other regulatory DNA sequences associated with RNAP III transcription (including DSE, STAF-binding site, Octamer element, SP1-binding site, PSE and B-box) may rather be considered as RNAP III transcriptional enhancer elements. Accordingly, the function of PTF/SNAPc (or possibly some of the subunits of this protein complex) resembles more that of a transcriptional activator. Likewise, individual proteins (modules) within TFIIIC may primarily be regarded as general transcription factor (τA) or as activator/co-activator (τB), respectively. In that sense, TFIIIC may be comparable to the RNAP II transcription factor TFIID, which comprises not only the general transcription factor TBP, but also transcriptional co-activators, the TBP-associated factors (TAFs).^127,128 In addition, in vitro transcription has helped to shed light on the regulation of RNAP III transcription by activators, co-activators and repressor proteins. In summary, in vitro transcription has not only been decisive for the identification of the proteins required to reconstitute basal and activated human RNA polymerase III transcription, but also crucial for defining minimal RNAP III promoters in eukaryotes. Collectively, past and very likely also future results obtained by in vitro transcription have provided and will continue to provide important information for understanding the mechanisms underlying human RNAP III transcription. Therefore, in vitro transcription assays will remain irreplaceable to complement our understanding of the regulation of RNAP III transcription that will be determined in more physiological systems in the future.