Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

Abstract

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inf lorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain Ϸ30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.Eukaryotic nuclear RNA polymerases interact with transcription factors to recognize gene promoters and initiate transcription. Many of the required factors can be purified individually and added back together to reconstitute transcription in vitro, allowing their identification and molecular characterization (1-7). However, proteins not required under a given set of assay conditions can be overlooked, though they may play important roles in vivo. The latter insight has come, in part, from the characterization of RNA pol II holoenzyme complexes in yeast and mammals (8)(9)(10)(11)(12). These complexes are several megadaltons in size and include pol II core enzyme subunits, most of the general transcription factors, and a growing list of additional activities including protein kinases (13), chromatin remodeling activities(14), and proteins involved in DNA repair (12).There has been at least one report of a pol I-containing fraction from rat cell-free extracts sufficient for accurate transcription in vitro (15), and several studies have shown that pol I transcription factors interact. For instance, the vertebrate pol I transcription factors UBF and SL1͞TIF-IB interact both in solution and on the DNA (16-18). Likewise, UBF and pol I cosediment in glycerol gradients and interact on Far-Western blots (19). Two additional factors, TIF-IA and TIF-IC copurify with pol I on multiple columns (20,21). Collectively, these results suggest that the pol I transcription factors might be associated with one another, possibly within a complex analogous to the pol II holoenzyme.Encouraged by brief reports that cell-free rRNA gene transcription could be achieved in plant extracts (22, 23), we set out to develop a cell-free system from broccoli (Brassica oleracea) to further our studies of rRNA gene regulation in Brassica and Arabidopsis (24-28). We show that a pol Icontaining activity sufficient for pr...

Multiple interactions between RNA polymerase I, TIF‐IA and TAF I subunits regulate preinitiation complex assembly at the ribosomal gene promoter

“…TIF-IA has been shown to be part of the Pol I 'holoenzyme', the enzyme moiety that is associated with most, if not all, factors required for transcription initiation (Saez-Vasquez and Pikaard, 1997;Seither et al, 1998;Albert et al, 1999). Moreover, TIF-IA has been shown to interact with TIF-IB/SL1 (Miller et al, 2001), which suggests that TIF-IA bridges both protein complexes.…”

Mechanisms of genomic rearrangements and gene expression changes in plant polyploids

“…(78,79) Transient and in vitro transcription assays have shown that Arabidopsis and Brassica rRNA promoters can function with the RNA Polymerase I transcription machinery of the other species. (78,80) Silenced rRNA genes in Arabidopsis and Brassica allotetraploids were reactivated by chemical inhibitors for DNA methylation and/or histone deacetylation, suggesting that rRNA genes are silenced by DNA and histone modifications presumably associated with inactive chromatin structure. (81) Collectively, the data suggest that rRNA gene silencing acts on chromosomal loci that result in cooperative silencing of rRNA genes.…”

“…Consistent with this notion, the enhancer imbalance model (76) suggests that, in Xenopus interspecific hybrids, dominant rDNA clusters have stronger enhancers that titrate the available transcription factors so that the rDNA clusters with weaker enhancers are inaccessible to the transcription activators and are not transcribed. Although this model is not supported by transient and in vitro and in vivo transcription assays in plants, (78,80) the involvement of species-specific factors should not be ignored. These factors may represent upstream regulators in a regulatory pathway.…”

Characterization of AtNUC-L1 Reveals a Central Role of Nucleolin in Nucleolus Organization and Silencing of AtNUC-L2 Gene in Arabidopsis

“…Primer tis maps the transcription initiation site accurately, TIS (Figure 9C, lanes 5-7;Saez-Vasquez and Pikaard, 1997) and allows quantification of pre-rRNA precursors, whereas primer p accurately maps the primary cleavage site, P (Figure 9C, lanes 11-13;Saez-Vasquez et al, 2004) and allows quantification of processed pre-rRNA precursors. To compare the ratio between primary pre-rRNA and cleaved prerRNA (TIS/P) in WT and Atnuc-L1 plants, tis and p primers were added simultaneously to the same primer extension reactions ( Figure 9C, lanes 8 -10).…”