Unusual enhancer function in yeast rRNA transcription.

Abstract

The rRNA genes in most eucaryotic organisms are present in a tandem array. There is substantial evidence that transcription of one of these genes may not be independent of transcription of others. In particular, in the yeast Saccharomyces cerevisiae, the enhancer of rRNA transcription that lies 2.2 kilobases 5' of the transcription initiation site is at least partly within the upstream transcription unit. The transcription by RNA polymerase II of a wide variety of genes is profoundly influenced by cis-acting short-nucleotide sequences called enhancer elements that can lie upstream or downstream of the transcription unit, at a distance of up to several kilobases. In many cases, the enhancer elements bind protein factors that are necessary for enhancer function. Although a number of models have been proposed (reviewed in reference 30), a clear understanding of enhancer function is not at hand.Two features distinguish rRNA genes from other genes in the eucaryotic cell. They are transcribed by a specific enzyme, RNA polymerase I, and they are arranged in a tandem array. This unique genetic structure has led to speculation that transcription of an individual gene is not independent of transcription of the neighboring genes; e.g., termination at one transcription unit may lead directly to initiation at the neighboring downstream promoter (7,12,15, 26).We have identified a sequence element, specific for RNA polymerase I, that has many of the characteristics of an RNA polymerase II enhancer (9, 10). In this paper, we explore aspects of the function of RNA polymerase I and its enhancer to ask about the parallels between polymerase I and polymerase II enhancers and about the importance of the tandemness of rRNA genes to polymerase I activity.Transcription by RNA polymerase I has been reviewed recently (38). The spacers between individual rRNA transcription units vary in length from 2 kilobase pairs (kb) (yeasts) to >30 kb (mammals). Within them are found a variety of sequence elements that influence the level of transcription. In Xenopus laevis, for example, these include several copies of a sequence closely related to the promoter, dubbed spacer promoters (6,7,22,23,29,33), each followed by about 10 copies of a 60-to 81-base-pair (bp) repeat, that behave in some respects like enhancer elements (22,23,29). The spacer promoters are curious because although they can give rise to transcripts and can stimulate transcription from an adjacent real promoter (6,7,29), the transcription from the spacer promoter seems not to be involved in this stimulation (7,25). Repetitive elements, some related to the promoter, have also been found in the nontranscribed spacer * Corresponding author. t Present address:

Yeast RNA Polymerase I Enhancer Is Dispensable for Transcription of the Chromosomal rRNA Gene and Cell Growth, and Its Apparent Transcription Enhancement from Ectopic Promoters Requires Fob1 Protein

“…These features are orientation independence, action from either upstream or downstream of the reporter gene, and action over long distances (more than 2 kb from the promoter), which are quite different from known yeast enhancers for Pol II. In addition, using the Pol I reporter integrated at the URA3 locus, it was observed that transcription of the reporter gene increases roughly in proportion to the number of enhancer elements (10). These observations are highly consistent with the model proposed here that the enhancer element increases the probability of localization of the reporter gene to be near the nucleolus, perhaps through its interaction with rDNA, rather than by stimulating Pol I transcription directly.…”

“…Northern analysis of RNA and Southern analysis of DNA were done by standard procedures (27). T7 reporter transcripts from plasmids YCprR8 (rR8) and YCprR10 (rR10) were detected with a 32 P-labeled antisense riboprobe prepared with pSP-T7(ϩ) by the method of Johnson and Warner (10). Analysis of the 5Ј end of 35S rRNA by primer extension was performed by using a 32 P-labeled primer which hybridizes to the 35S rRNA external transcribed spacer as described previously (36).…”

“…In fact, we have found that without some special mechanism, which apparently requires both the enhancer and the Fob1 protein, transcription from this ectopic Pol I promoter is extremely inefficient. It was previously observed that the effect of the enhancer was higher when the T7 Pol I reporter was integrated at the URA3 locus compared to the effect observed in the rR8-rR10 plasmid system; as much as a nearly 100-fold stimulation of Pol I transcription by the enhancer was reported for the integrated reporter system (10). The Pol I promoter on rR8 without the enhancer appears to have a limited but higher accessibility to the Pol I machinery in the nucleolus than the same reporter integrated at URA3.…”

Efficient termination of transcription by RNA polymerase I requires the 5′ exonuclease Rat1 in yeast

“…Our ChIP data localized Rat1 to both IGS1 and the 5Ј region of the 35S rDNA, possibly reflecting looping of the rDNA as proposed previously (Johnson and Warner 1989;Kulkens et al 1992). This might arise through oligomerization of Reb1, since this binds the rDNA at T1 in IGS1 and just upstream of the Pol I promoter in IGS2 (Reeder and Lang 1997).…”

The rRNA enhancer regulates rRNA transcription in Saccharomyces cerevisiae.

“…Its effect is relatively independent of position and orientation (10). Unlike enhancers of RNA polymerase II in S. cerevisiae, this enhancer is active even when downstream of its target gene (15).…”