TRENDS in Plant Science Vol.6 No.3 March 2001
RNA polymerase I holoenzymes Alexander Kenzior and William Folk In eukaryotes, holoenzymes are large preassembled complexes containing RNA polymerases and variable sets of general transcription initiation factors and cofactors that are important for the regulation of gene expression. Recent advances in purification and characterization of RNA polymerase I holoenzyme from plants provide experimental data suggesting that it plays a key role in transcriptional regulation. These findings have a significant implication on our understanding of the mechanisms of promoter recognition, assembly of transcription initiation complexes, RNA chain elongation and transcription termination.
The considerable progress recently achieved in our understanding of the eukaryotic transcription machineries has relied upon their accessibility from yeast, Drosophila and animal cells. By contrast, the isolation of the plant transcription machineries has proven to be less tractable; however, a notable exception occurs with the Pol I machinery of Brassica oleracea, where significant advances have been made and there is the promise of many more1,2. Promoters
In plants, as in most eukaryotes, rRNA genes are clustered in one or several chromosomal sites as head-to-tail tandem repeats. Much of our understanding of the structural and functional organization of eukaryotic rRNA genes, including plants, comes from pioneering studies of the organization of rRNA genes of Xenopus laevis3,4 (Fig. 1). Between each tandemly repeated rDNA coding sequence are spacers that contain the rRNA gene promoters, enhancers and sequences required for transcription termination. The region immediately upstream of the initiation site (+1) is necessary for initiation of basal transcription. It contains the only conserved rRNA promoter sequence element, the ribosomal initiator (rInr), an AT-rich TATA-like sequence surrounding the initiation site. This element mediates correct but inefficient initiation: the
remainder of the promoter is bound by a core transcription factor. For many years, the predominant view of the role of this factor was that it recruits RNA polymerase I in a stepwise assembly process3. Additional elements and factors help to assemble and stabilize the transcription complex formed on the core promoters of yeast and animals (yet to be shown for plants). The upstream promoter element (UPE) that extends 150 –200 bp upstream of the initiation site significantly stimulates transcription, as does the transcriptional terminator of the preceding rRNA repeat. The upstream terminator serves several functions, including protection of the promoter from wandering polymerases, remodeling of chromatin over the promoter and possibly folding of the rRNA repeats in the nucleolus3.
Purified eukaryotic RNA polymerases are structurally distinct multisubunit enzymes that, until recently, had been thought to resemble the core enzymes of bacterial RNA polymerases. The prevailing view of transcription initiation posited an ordered, stepwise assembly of transcription factors on a promoter, which then was bound by the core polymerase (either polymerase I, II or III). This view began to change with the discovery that transcription initiation by RNA polymerase II can involve a preassembled holoenzyme complex containing Pol II and the general transcription factors and other subunits and activities, and that much of the functional RNA polymerase activity within a cell was present as a holoenzyme5,6. Subsequent studies have suggested that the other two RNA polymerases also form holoenzymes. Holoenzymes
Much of our early thinking about RNA polymerases derives from studies of prokaryotes, where a core enzyme – a catalytically active RNA polymerase that binds DNA and substrate – is competent for RNA chain elongation but is unable to specifically recognize promoters. Additional initiation-specific subunits are required to form an autonomously initiating entity – the holoenzyme.
Given the evolutionary conservation of general mechanisms of transcription initiation by all three classes of nuclear RNA polymerases, is it reasonable to assume that similar features are shared by the pol I and pol III transcription machineries? Evidence for an RNA polymerase III holoenzyme was first reported more than a decade ago7 and confirmed by the isolation and characterization of a pol III holoenzyme.
18S 113 bp repeats
18S Defective terminator
60/81 Repeats (enhancers)
Terminator TRENDS in Plant Science
Fig. 1. Arrangement of the gene promoter, spacer promoter duplications and repetitive sequence elements within the rDNA intergenic spacers of (a) Arabidopsis thaliana and (b) Xenopus laevis.
http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01887-8
It contains the essential transcription factors TFIIIB and TFIIIC, and is capable of accurate transcription of tRNAs and other class III genes8. Strong evidence for a pol I holoenzyme has come from several sources, including extensive purification of a pol I containing cell-free transcription system from broccoli (B. oleracea) inflorescence in Craig Pikaard’s laboratory2,9–11. The extensively purified plant pol I transcription holoenzyme programmed transcription initiation of naked DNA from the in vivo start site and used the same core promoter sequences required in vivo. Approximately two to ten percent of pol I might be present as a holoenzyme complex (molecular mass ~1.7 MDa), which contains ~30 distinct polypeptides. Because plant pol I transcription factors have not yet been cloned and characterized, and only a few subunits of the plant pol I core enzyme have been cloned, identification of proteins in the putative pol I holoenzyme is limited to the A190, AC19 and the A27 subunits of the pol I enzyme, for which antibodies are available. RNA polymerase I multimeric complexes from X. laevis, and murine and rat cells have also been purified and partially characterized. The X. laevis holoenzyme was purified to nearhomogeneity using the same steps employed for the plant holoenzyme. It contains ~55 proteins including the TATA binding protein, casein kinase II and histone acetyltransferase activities11; casein kinase II and histone acetyltransferase activities have also been documented in the plant holoenzyme preparation. The identification of RNA polymerase I holoenzymes raises provocative questions about their mode of template binding. Several alternative possibilities exist, including: • A concerted reaction in which transcriptionally competent holoenzyme complexes are recruited to the promoter in a single DNA-binding event. • A stepwise reaction in which transcription factors are recruited to a promoter before RNA polymerase, and thereby facilitate the association of holoenzyme into the transcription complex. • Independent recruitment of transcription factors and holoenzyme. http://plants.trends.com
TRENDS in Plant Science Vol.6 No.3 March 2001
DNase footprinting has revealed that one or more proteins present in the Brassica holoenzyme interact with the rRNA gene core promoter region. Agarose Electrophoretic Mobility Shift Assay analysis has shown that promoter-specific binding activity copurifies with promoterdependent transcription activity. Titration, time course and competition analysis have revealed the formation or dissociation of a single protein–DNA complex that can be detected by virtue of the formation of nascent RNA transcripts, indicating that complexes contain RNA polymerase I (Ref. 1). Collectively, these data suggest that pol I transcriptionally competent preinitiation complexes assemble in a single DNA-binding event with the rRNA promoter. Additional questions might be raised about possible communication between the holoenzyme and proteins that help regulate initiation, elongation and termination of RNA transcription. The activity of the upstream binding factor that helps activate ribosomal RNA transcription is modified by phosphorylation and acetylation, and also, by binding of other proteins such as retinoblastoma protein12. Perhaps these are connected to the holoenzyme and are modulated by the casein kinase II and histone acetyltransferase activities? It would not be surprising if the termination factor that promotes release of transcripts and the polymerase3,13 was also to be a component of the holoenzyme. Additionally, the chromatin structure of the transcribed genes might be targets for activating or repressing activities present in the holoenzyme. Now that purified transcription machineries are available, we should be able to address these tantalizing suggestions.
4 Doelling, J.H. et al. (1993) Functional analysis of Arabidopsis thaliana rRNA gene and spacer promoters in vivo and by transient expression. Proc. Natl. Acad. Sci. U. S. A. 90, 7528–7532 5 Thompson, C.M. et al. (1993) A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast. Cell 73, 1361–1375 6 Greenblatt, J. (1997) RNA polymerase II holoenzyme and transcriptional regulation. Curr. Opin. Cell Biol. 9, 310–319 7 Wingender, E. et al. (1986) Association of RNA polymerase III with transcription factors in the absence of DNA. J. Biol. Chem. 261, 1409–1413 8 Wang, Z. et al. (1997) Identification of the autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition. Genes Dev. 11, 2371–2382 9 Seither, P. et al. (1998) Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors. J. Mol. Biol. 275, 43–53 10 Hannan, R.D. et al. (1999) Identification of mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system. Nucleic Acids Res. 27, 3720–3727 11 Albert, A-C. et al. (1999) Histone acetyltransferase and protein kinase activities copurify with putative Xenopus RNA polymerase I holoenzyme selfsufficient for promoter dependent transcription. Mol. Cell. Biol. 19, 796–806 12 Pelletier, G. et al. (2000) Competitive recruitment of CBP and Rb-HDAC regulates UBF acetylation and ribosomal transcription. Mol. Cell. 6, 1059–1066 13 Jansa, P. and Grummt, I. (1999) Mechanism of transcription termination: PTRF interacts with the largest subunit of RNA polymerase I and dissociates paused transcription complexes from yeast and mouse. Mol. Gen. Genet. 262, 508–514
Alexander Kenzior William Folk* Dept of Biochemistry, University of Missouri – Columbia, Columbia, MO 65211, USA. *e-mail: [email protected]
Contribution from the Missouri Agricultural Experiment Station. Journal Series Number 13120. References 1 Saez-Vasquez, J. and Pikaard, C.S. (2000) RNA polymerase I holoenzyme-promoter interactions. J. Biol. Chem. 275, 37173–37180 2 Saez-Vasquez, J. and Pikaard, C.S. (1997) Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiate rRNAgene transcription in vitro. Proc. Natl. Acad. Sci. U. S. A. 94, 11869–11874 3 Paule, M.R. and White, R.J. (2000) Transcription by RNA polymerases I and III. Nucleic Acids Res. 28, 1283–1298
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