The regulation of rRNA synthesis is intimately tied to regulation of cell growth rate, and synthesis of rRNA by RNA polymerase I is the major transcriptional activity of the cell, accounting for 60% of total transcription in rapidly growing yeast cells (56
). In spite of this critical role, certain aspects of Pol I transcription are still poorly understood (reviewed in references 12
, and 56
). The situation is complicated by the fact that the genes encoding rRNA are multicopy. Thus, rRNA synthesis could be modulated by varying the transcription rate per gene or by varying the number of active genes (1
). Electron microscopy (EM) visualization of active rRNA genes from many different cell types by the Miller spreading method typically shows genes rather heavily packed with polymerases and nascent rRNA transcripts in the familiar Christmas tree configuration (27
). Since these active rRNA genes are highly transcribed, it appears, that once a gene is open for transcription, there is no shortage of Pol I for initiation. Based largely on EM observations, two recent review articles have indeed concluded that Pol I transcription does not appear to be limited by initiation rate (30
). There is additional direct support for this idea from other EM studies (24
). For example, polymerase density is uniformly high on any rRNA gene that is activated after introduction into Xenopus
oocytes, even when the introduced genes are in large excess over endogenous genes (38
). These observations and others led to a model in which initiation-competent Pol I is present in excess in actively growing cells and in which rRNA synthesis is regulated by controlling the number of active genes (43
). In this model, each gene is a “binary unit” that is either on or off and, if on, is producing rRNA at approximately the same rate as other active genes due to high Pol I loading.
Other evidence besides EM is consistent with rRNA transcription being regulated by changing the number of active genes. Exponentially growing cells use only half or fewer of their total complement of rRNA genes (6
), and it has been shown in mammalian and budding yeast cells that the number of active genes decreases when cells undergo the transition from log to stationary phase (7
). It was recently demonstrated that this gene inactivation in yeast (as determined by accessibility to psoralen cross-linking) is dependent on the histone deacetylase Rpd3 (46
). In mammalian cells, the nucleolar remodeling complex, NoRC, recruits HDAC1 and DNA methyltransferases to inactive rRNA gene repeats (48
). Combined with reports showing various correlations between chromatin modifications and the activity level of rRNA genes (e.g., references 16
, and 52
), it is clear that epigenetic mechanisms either control or enforce the ratio of active to inactive genes. In NIH 3T3 cells, chemical inhibitors of DNA methylation and histone deacetylation result in an increase in endogenous rRNA transcription (16
). One interpretation of these findings is that the number of active genes, and not initiation by Pol I, is limiting for rRNA transcription, consistent with the model based on EM visualization of genes.
On the other hand, a large body of evidence points to a specific Pol I transcription factor, Rrn3p in yeast or its mammalian homologue hRrn3 or TIF-1A, as being the growth regulatory factor for rRNA synthesis (3
). This protein functions by binding directly to Pol I (20
) in an interaction controlled by phosphorylation (5
) and renders Pol I competent for initiation (25
), apparently by bridging the interaction between Pol I and other components of the preinitiation complex (26
). Only a small fraction of both Pol I and Rrn3p is in the active complex, and the active Pol I-Rrn3p complex has been shown to be limiting for rRNA transcription in stationary or growth-arrested cells (3
). Thus, at least during down-regulation, Pol I transcription is regulated by the concentration of the Pol I-Rrn3p complex and thus by initiation rate, rather than by the number of active genes. As predicted, this results in a lower average polymerase density on active rRNA genes in post-log-phase cells (46
). What is not known is whether the concentration of the active Pol I-Rrn3p complex is also limiting for transcription in exponentially growing cells. A clear prediction of the binary-unit model is that the active Pol I-Rrn3p complex is present in excess in logarithmically growing cells such that the number of genes open for transcription is limiting.
We addressed this question in exponentially growing Saccharomyces cerevisiae
cells. A published observation seemed difficult to reconcile with the prevailing model, in which each gene is a binary unit of activity. That is, experiments in yeast cells showed that it was possible to decrease the total number of rRNA genes from the typical number of ~150 to ~40 without affecting cell growth rate or rRNA synthesis rate (21
). Since psoralen cross-linking assays indicate that about half of the total number of genes (or ~75 genes) are active in wild-type yeast cells (7
), the prediction of the binary unit model would be that wild-type cells with ~75 active rRNA genes would have a higher rRNA synthesis rate than cells with only ~40 rRNA genes total. However, given uncertainties about the psoralen cross-linking assay discussed elsewhere (2
), such as the possibility that a psoralen-accessible gene may be in an open chromatin configuration but not yet transcribed, it remained possible that the number of transcribing genes was regulating rRNA synthesis. We undertook a new approach to address this long-standing question. We directly counted the number of active genes per nucleolus and the number of polymerases per gene. When gene number was reduced, the available polymerases loaded with a higher density on fewer genes to produce the same amount of rRNA as in control cells. On some genes in the reduced copy strain, there was a polymerase every 41 nucleotides (nt), which is near the limit possible for polymerase packing. Our results show that rRNA synthesis in exponentially growing yeast cells is controlled by the ability of cells to load polymerases and not by the number of open genes.