RNA polymerase

III transcription

Alan P. Wolffe National

Institutes

of Health,

Bethesda,

Maryland,

USA

Remarkable progress has been made in defining the functional significance of the protein-DNA interactions involved in transcription complex formation on yeast tRNA and 55 RNA genes. This new information leads to a re-evaluation of how the class III gene transcription machinery operates.

Current

Opinion

in Cell Biology

Introduction Much of the current doctrine concerning the mechanism and regulation of eukaryotic gene expression has been established using class III genes as model systems. Concepts that were innovative when developed with 5s RNA and tRNA genes have now become commonplace. Among these we might include: the discovery of regulatory elements within genes; the requirement for transcription factors to assemble complexes capable of recognition by RNA polymerase; the stable sequestration of transcription factors onto and around the gene regions; competition between transcription factors and the histone proteins for binding to the gene; and the differential stability of transcription complexes [ 1,2]. The last 2 years have seen a great deal of continued progress in understanding the mechanism and regulation of class III (C) genes. The purpose of this review is to illustrate the concepts that have emerged, and to discuss their implications for eukaryotic gene expression in general. Pre-eminent among the new ideas brought forward is the demonstration that the key class III gene transcription factor (TFIIIB) has its DNA-binding potential activated by other proteins, that it is directed into position by these proteins and that it binds DNA tightly, but without specificity. Much evidence has also been presented suggesting a great deal of mechanistic similarity between the transcription of class II and class III genes. Final&, a substantial series of experiments has described molecular mechanisms that might regulate class III transcription in viva, including transcription complex stability and tmnscription factor activity and abundance [l-4,5**,&*].

The biochemical dissection transcription in yeast

Three transcription factors, TFIII& TFIIIB and TFIUC (also known a.5r) are involved in class III gene transcription. Three promoter elements have also been defined. The A-box is present in both tRNA and 5s RNA genes (Fig. la), and TFIIIC will bind to it when there is a B-box present in ~RNAgenes, or after TFIIIA has bound to the C-box in 5s RNA genes (Fig. lb). TFIIIB will only bind to a tRNA or 5s RNA gene if the binding of TFIIIC, or of TFIIIA plus T’FIIIC , has taken place (Fig. lc). RNA polymerase III recognizes the transcription complexes once TFIIIB has bound; however, promoter strength is further inIluenced by 5’-flanking sequences (Fig. Id). Geiduschek and colleagues [3,4,5**,6m*] were able to demonstrate the functional signkicance of the proteinDNA interactions documented in Fig. 1 by controlling the availability of RNA precursors. The limited elongation of RNA polymerase III under these conditions allowed the entire transcription initiation process to be described. As shown in Fig. l(e), TFIIIB remains associated with the upstream regions of the gene, in spite of RNA polymerase III initiating transcription and elongating through the gene, away from the start site of transcription. The eventual fate of TFIIIA and TFIIIC during the transcription process is less clear as these transcription factors are bound within the body of the 5s RNA gene, (or with TFIIIC alone, to the body of the tRNA gene). In fact, the exact locations of the various component polypeptides comprising TFIIIC along the internal promoter of the 5s RNA gene has been mapped and earlier work suggests that TFIIIA and TFIIIC can stay in place during the transcription process as indicated in Fig. l(e) [7]. It was not clear, however, whether the continued presence of TFIIL4 and TFIIIC is required for transcription to proceed. A dramatic solution to this question was obtained through the selective dissociation of these proteins from the gene

[5-l.

of class III gene

An important series of papers have appeared describing in detail the composition and stepwise assembly of transcription complexes on Saccbarom~es cerevtie 5s RNA and tRNA genes [3,4,5 l , 6-I. This process is represented in Fig. 1.

@ Current

1991, 3:461-466

Biology

Transcription complexes assembled onto certain class III genes were known to be resistant to a variety of challenges, such as high salt, competitor DNA and dilution. Geiduschek and colleagues [3,4,5**,6*,] clearly demonstrated that the complex of TFIUB and the 5’-Ilanking region of a class III gene is resistant to high salt concentrations, the polyanion heparin (similar to competitor DNA)

Ud ISSN 0955-0674

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Fig. 1. A schematic representation of the transcription process for either a yeast tRNA gene or a yeast 55 RNA gene. (a) Three promoter elements have been defined: the A-box (present in both genes), the B-box and the C-box. (b) TFMC binds to the A-box in the presence of a B-box in the tRNA gene, and in the presence of TFIIIA bound to the C-box in the 55 RNA gene. (4 TFIIIB only binds to tRNA or 55 RNA gene if TFIIIC plus TFIIIA, respectively, are already bound. td) RNA polymerase Ill recognizes the transcription complexes once TFIIIB is bound. fe) TFIIIB remains associated with the upstream regions of both genes, even though RNA polymerase Ill, having initiated transcription, moves away from the start site of transcription. The eventual fate of TFIIIA and TFIIIC during transcription is not yet clear, but it seems that they remain bound within the promoter of the gene. (0 As in kc). The numbers in (a) refer to base positions at the extreme 5’ end of the DNA fragment f-50), the start site of transcription f + 1) and the end of the gene. The position of the various transcription factors on the promoter elements are only approximate.

RNA wlvmerase

and dilution. The first two treatments remove TFIIIC from a tRNA gene, and TFIIIA plus TFIIIC from a 5s RNA gene, respectively. TFIIIB alone remains bound to the 5’-flanking region under these conditions, and is still able to di rect multiple accurate transcription events by RNA polymerase III. The conclusion reached was that TFIIIB is the central transcription factor of yeast RNA polymerase III, and that TFIIIA and TFIIIC act as assembly factors, This conclusion has important general implications for eukaryotic gene expression. Specific DNA sequences within the 5’.flanking regions of class III genes are not essential for the efficient transcription of these genes, yet it is this region that TFIIIB interacts with. Moreover, TFIIIB itself is not a DNA-binding protein. This implies that TFIIIA and TFIIIC are not only essential for bringing TFIIII3 to the class III gene, but also for activating its DNA-binding activity and precisely positioning it at the appropriate place to interact with RNA polymerase III. It is remarkable that a transcription factor binds so tightly to DNA that it can only be dissociated by chaotropic agents, yet this binding is activated by protein-protein interactions and is non-specific. This elaborate process not only suggests the presence of many controls for actually establishing the active state (see later), but also implies that, once established, the TFIIIB-DNA complex will be difficult to disrupt and the subsequent repression of a gene hard to achieve. As 1will discuss later, this concept is hard to reconcile with the regulated expression of class III genes in ‘large eukaryotes’. However, if this observation is generally applicable, perhaps the well documented specific protein-DNA interactions described for many class II genes only serve to regulate the DNA binding of one key transcription factor. Once present, this key factor, which presumably interacts directly with RNA polymerase, renders the presence or absence of other DNA-binding transcription factors irrelevant. Considering the significance of this hypothesis, it is of considerable interest to examine other evidence supporting this model for class III genes.

In vivo veritas?

In vitro experiments with biochemical fractions always attempt to reconstruct what happens in the living cell. In reality, the biochemical reconstruction only provides possible pathways, and does not describe the actual molecular mechanism chosen by the cell. Reassurance that some approximation of the truth has been reached comes from making testable predictions or in vivo correlations. For the yeast class III gene transcription complexes, excellent in vivo correlations exist [ 81. Genomic footprinting of the 5’-flanking region of the S. cerevisiae tRNA gene revealed a region from - 40 (relative to the transcription start site) through to + 15 that was protected from DNase I cleavage. This corresponds to the expected DNA-protein interactions for TFIIII3 and RNA polymerase III (Fig. 1). Protection was also observed over the A- and B-box promoter elements and point

III transcrition

Wolffe

mutations within these boxes that reduced transcription in vitro eliminated the protection of the 5’4ankhg region. This implies that formation of the complex over the S-llanking region is at some stage dependent on protein-DNA interactions within the gene. The 5’-flanking region footprint changed in a manner that was dependent on growth conditions. When the gene was inactive the protection over the transcription initiation site disappeared, consistent with the absence of bound RNA polymerase III. We can conclude that the genomic footprinting and in vitro reconstruction experiments lead to the same conclusions regarding the organization of class III genes into transcription complexes.

Transcription eukaryotes

factors

from

yeast and larger

The first eukaryotic transcription factor to be purified and cloned was TFIIIA from Xenopus (reviewed in [ 21). Progress towards the purification and cloning of TFIIIA from other organisms, or of TPIIIC and TFIIIB from any organism, has been frustratingly slow. Clear data on the composition and properties of yeast TFIIIC have now been obtained [6**,9,10**,11,12]. The protein exists as a large complex of peptides ( > 300 kD molecular weight). Crosslinking studies suggest that distinct polypeptides exist of 145, 135 and 1OOkD molecular weight. The 145kD polypeptide interacts with the B-box, and the 135 and 1OOkD polypeptides interact with the A-box. In contrast to this remarkable consensus, comparable experiments in mammalian extracts lead to a diversity of opinions [1315]. However, the evidence for multiple components comprising TFIIIC appears compelling [ 13,141. There is clearly much to be learned from the cloning and characterization of the genes for the various component polypeptides of these transcription factors. Understanding why these simple genes require such complex transcription factors will yield important Insights into how class III genes, and perhaps also class II genes, are regulated.

A genetic approach transcription

to class III gene

An attractive approach to characterizing the genes encoding class III transcription factors and also the transcriptional role of the polymerase itself is to make use of the powerful tool of genetic analysis in yeast [16,17*]. In one specific yeast system, a tandem array of genes was created, such that an essential ~RNA gene (the ‘reporter’ gene) with no intrinsic promoter activity was transcribed by virtue of having immediately upstream another, nonessential tRNA gene (the ‘promoter’ gene) lacking an effective terminator. Thus, mutations in the genes encoding the transcription factor proteins could now be assayed in the transcriptional apparatus itself because the properties

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of the transcriotion factors and RNA nolvmerase could be monitored by assessing how well thky transcribed the ‘promoter’ gene and, consequently, the non-mutated ‘reporter’ gene. The promoter of the ‘promoter’ tRNA gene was crippled by mutation of the A-box. Eight dominant mutant strains of yeast were isolated following selection for expression of the ‘reporter’ gene, that could compensate for the A-box mutation in the ‘promoter’ gene. Preliminary results suggest that one mutant gene, PCFI, is probably a component of TFIIIC. A similar selection procedure led to the suggestion that alterations in RNA polymerase III itself affect the efficiency of transcription termination. These are exciting approaches to the mechanistic basis of class III gene transcription and much more definitive progress can be expected in the future.

Regulating

class III gene transcription

In spite of the evident superiority of yeast for use in dissecting the actual mechanistic basis of class III transcription and for purifying and cloning TFIIIA, TFIIIB and TFIIIC, larger eukaryotes have the advantage that their class III genes are known to be regulated in interesting ways. Donald D. Brown and I [2] have proposed that diIferences in the stability of class III gene transcription complexes can account for the differential regulation of these genes during Xenopus development. The interaction of TFIIIC and TFIIIA with individual 5s RNA genes determines transcription complex stability and, consequently, whether particular genes will remain active or be repressed [W-20**]. If a stable transcription complex is assembled on a gene, that gene remains active; if not, the gene is repressed. The significance of these experiments, which again attempt to reconsuuct a developmental switch in a test tube, stems from in vivo correlations that clearly demonstrate the importance of both the A- and C-boxes of the 5s RNA gene, i.e. the site of interaction of TFIIIA and TFIIIC with the gene, and of the concentration of TFIIIA in regulating transcription during embryogenesis. Importantly, the transcription systems used in vitro are very efficient and conditions can be established in which essentially all the genes are active and transcription complex footprints similar to those seen in yeast can be obtained [21=*]. TFIIIA and TFIIIC also rapidly interact with 5s RNA genes, whereas TFIIIB binding is slow. These proteins will therefore compete with the histone proteins for binding stably to the Aand C-boxes [22*-24.1. If TFIIIA and TFIIIC bind stably, histones will be excluded, whereas if they do not, nucleosome formation will repress transcription. These results are hard to reconcile with the properties of the transcription factors within the yeast transcription complex, in particular the stable binding of TFIIIB. However, transcription factors of larger eukaryotes are known to bind to class III genes with different protein-DNA interactions and~tiesfromthoseinyeast(raiewedin [l]). viral infection of mammalian cells leads to regulated changes in class III gene transcription. Both adenovirus ElA protein and the hepatitis B virus X-gene product

will activate transcription of class II and class III genes [ 25,26**]. In both instances, extracts from infected cells synthesize RNA efficiently at a high template concentration which normally inhibits transcription. This observation provides indirect evidence for an Increase in either the quantity or activity of a limiting transcription factor [ 27,281. Kinetic measurements of the rate and amount of stable complex formation suggest that TFIIIC is the factor affected. How an increase in the abundance or activity of TFIIIC is accomplished remains to be determined. Class III genes are also regulated during mouse embryogenesis [29]. The B2 genes are rodent-speciIic middle repetitive elements transcribed by RNA polymerase III. They are expressed in the ectoderm and mesodenn, but not in the embryonic or extra-embryonic endoderm of early embryos. This tissue specificity is mimicked in vitro by an embryonal carcinoma cell line as F9 [30]. This cell line can be induced to differentiate into endodermlike cells in response to various agonists. Nuclear run-on experiments show that the downregulation of B2 genes during F9 embryonal carcinoma cell differentiation occurs at the transcriptional level. All class III genes show a similar inhibition of transcription, which is due to a reduction in TFIIIB activity. This result is similar to that of converting log phase mammalian or yeast cells to stationary phase, leading to a concomitant reduction in class III gene transcription [31]. AU of these experiments have yet to resolve whether limitation of TFIIIC and/or TFIIIB activity is a consequence of changes in post-translational modification or in the abundance of a particular transcription factor, or is due to the alleviation of an inhibitory activity. Production of antibodies and the cloning of cDNAs encoding the various polypeptides comprising these transcription factors will be required before definitive answers appear.

Hybrid

transcription

There is a growing body of evidence imp!ying an evolutionary relationship between the class II and class III gene transcriptional machinery. Certain class III genes are predominantly controlled by regulatory elements that lie in the 5’-flanking region and that are similar to those regulating class II genes [32,33**,34]. Although there is some disagreement about the relative importance of particular elements and their cognate transcription factors, there is general agreement that some transcription factors are used in the transcription of both class II and class III genes. Two of these hybrid genes with properties between those of class II and class III genes encode ~6 and 7SK RNA The promoters of these genes contain a sequence, about 30 bp upstream of the start site of transcription, that is similar to the TATA box and which is required for efficient transcription by RNA polymerase III. Purified yeast TFIID, which interacts with this TATAlike box is able to direct transcription initiation by RNA polymerase III [35**]. This capacity of RNA polymerase III to use a transcription factor implicated in basal class II gene

RNA polymerase

transcription parallels the close structural relationship between RNA polymerase II and III [36].

III transcription

Wolffe

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AcknowledRements I thank Randall Morse for reading the manuscript and Thuy Vo for preparing it. I apologize to my colleagues for not being able to comprehensivety highlight their important contributions before 1990.

Overview

and prospects

for the future

Class III genes have many advantages associated with their simplicity. Every conceivable mutation has been in traduced into almost every type of these genes in the past decade. This has facilitated the delinition of regulatory elements and the transcription factors that interact with these elements. Eflicient in vitro transcription systems encouraged the hope that transcription factors would soon be fractionated and put-&d. In yeast, at last, this hope has been realized. The tour de force from Geiduscheck’s laboratory [3,4,5**,6**] in characterizing the transcription complexes of certain yeast tRNA and 5S RNA genes has once again established major interest in class III gene transcription. Many questions are now raised. Are TPIIIA and TPIIIC merely assembly factors and how is the DNAbinding capacity of TPIIIB activated. What is the nature of the incredibly stable interaction of TPIIIB with DNA and how does RNA polymerase III recognize TPIIB? If TPIIIC is merely an assembly factor, why does it have such a complex organization? Are these observations general to all yeast genes? Why do the transcription complexes of large eukaryotes behave somewhat differently? The next few years should bring the long-awaited cloning of cDNAs encoding some of the polypeptide chains of the different transcription factors after which insights into these problem should follow in abundance. It is important to bear in mind the caution that work with yeast may not be wholly applicable to larger eukaryotes, although we hope that the molecular mechanisms employed in class III transcription in different organisms will have a common evolutionary origin and will therefore have found common solutions to the problem of regu lating gene expression. It would appear that this is the case, especially if mechanistic details are conserved between class II and class III genes. The evidence reviewed here suggests that there are many similarities, but also differences, between yeast and larger eukaryotes. Particularly relevant is the question of class III gene regulation, a well documented phenomenon in large eukaryotes Secting examples of each gene type as well as all transcription factors. We do not know if similar mechanisms operate in yeast or indeed whether yeast needs to regulate its class III genes in any comparable way. Much of the work with class III genes in larger eukaryotes is phenomenological: different extracts are prepared and fractionated, and properties of the transcription systems are then assessed. It is to be hoped that the development of antibodies to some of the more purified transcription factor preparations will lend more rigor to the published observations. The purification and characterization of the yeast transcription factors must surely help in this respect. Class III gene transcription has raised many questions, and we can expect biology to have many surprising solutions in prospect.

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TFIIIA Alone, Prevents NucleosomaI Repression of Transcription. J Biol @em 1990, 265:5014-5023. A complete transcription complex resists nucleosome-mediated repres sion of transcription much more e5ectively than TFIIIA alone. Histones H3 and H4 do not repress uanscription if pre-assembled onto 5s DNA whereas histones H3/H4/H2A and H2B do. PATEL G, JONES NC: Activation In vffm of RNA PoIymerase 25. II and III Directed Transcription by BacuIcvirus Produced ElA Protein. Nucleic Acti Res 1990, 18:290+2915. 26. ALIFIERO B, SCHNEIDER RJ: The Hepatitis B Virus X-Gene .. Product Trans.Activates Both RNA Polymerase II and III Promoters. EMBO J 1990, 9:497-504. The activation of class III gene transcription in a mammalian cell following hepatitis B virus infection is explained by an increase in TFIIIC actMy mediated by the hepatitis B virus X-gene product. 27.

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AP Wolffe, L&oratory of Molecular Embryology, MCI-ID, Building 6, Room

USA

131,

National

Institutes

of Health,

Bethesda.

Maryland

20891,