Visual Analysis of the Yeast 5S rRNA Gene Transcriptome: Regulation and Role of La Protein

Abstract

5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, ϳ132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of ϳ1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.In eukaryotes, nuclear RNA transcription is carried out by one of three specialized RNA polymerases, Pol I, II, and III, with Pol III dedicated to making short, untranslated RNAs, including tRNAs, 5S rRNA, and many others (12). Consistent with the central role of these RNAs in protein synthesis, Pol III is an essential enzyme whose activity is regulated by various growth control pathways and is deregulated in human cancer cells and during cardiac hypertrophy (23, 67). Many years of study have led to detailed knowledge of the different types of promoters and of the trans-acting factors involved in Pol III gene expression (20,31,56). For the Saccharomyces cerevisiae 5S RNA genes studied herein, three factors are required: TFIIIA, which specifically binds the internal promoter of 5S genes, and TFIIIB and C, which are required for all yeast Pol III genes. Preinitiation complex formation begins with the deposition of TFIIIA on the internal promoter, followed by recruitment of the six-subunit TFIIIC complex, which contacts TFIIIA as well as sequences within the gene (Fig. 1A). TFIIIC recruits the three-subunit TFIIIB complex, which binds a conserved upstream regulatory sequence and is necessary and sufficient for Pol III transcription (30). TFIIIB, which contains TATA binding protein, recruits Pol III to the promoter and plays an essential role in the formation of the open initiation complex. The stability of TFIIIB, which restructures the DNA and recruits Pol III through multiple rounds of transcription (7,30), renders these genes very highly transcribed and the most prominent binding sites for TATA binding protein in the cell (51). The internally bound factors do not form a barrier to Pol III elongation.We used Miller chromatin spreading (39) to directly visualize the 5S gene population in actively growing yeast cells. This electron microscopy (EM) method has frequently been applied to the study of Pol I and Pol II gene expression. Pol III genes, h...

Transcription termination by nuclear RNA polymerases

“…Other in vitro studies show no role for La protein in RNAPIII transcription (Pannone et al 1998;Weser et al 2000;Hu et al 2003). Recent work from French et al (2008) might reconcile both hypotheses. Using EM to visualize RNAPIII on the yeast 5S rRNA, a higher average RNAPIII density was detected in a strain deleted for the yeast La protein, and ChIP assays demonstrated an association of La protein with the 5S gene, apparently mediated by the nascent RNA.…”

“…Using EM to visualize RNAPIII on the yeast 5S rRNA, a higher average RNAPIII density was detected in a strain deleted for the yeast La protein, and ChIP assays demonstrated an association of La protein with the 5S gene, apparently mediated by the nascent RNA. French et al (2008) showed that La has no effect on the rate of synthesis and size of 5S rRNA but proposed that the extra RNAPIII visualized at the 5S gene in the yeast laD strain reflects a defect in RNAPIII release.…”

The Paf1 complex is required for efficient transcription elongation by RNA polymerase I

“…The reason for positive ChIP signal in the regions of rDNA that are devoid of Pol I remains unknown. However, it is known that Pol II transcripts originate from the NTS regions of the rDNA, and Pol III is active at the 5S rRNA genes (27,28). Thus, this signal may result from association of these factors with the other polymerases that are clearly active in the spacers between 35S rRNA genes.…”

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation

“…On the genes with a hairpin ≥ 5 nt upstream of the T-tract, RNAP III would have to back up as part of termination but, as shown by the new data, without RNA 3′ end retraction. 13 The elongation rate for RNAP III on yeast 5S rRNA genes has been calculated to be 60-75 nt/s with an occupancy of 2-3 RNAP III molecules per 5S gene and a reinitiation interval of 1.2 s. 94 Calculations based on tRNA levels in yeast estimate the rate of RNA production, i.e., termination, at 2-4 transcripts/gene/s 95 (or a time between two successive terminating RNAP III molecules equal to 250-500 ms). To put it another way, an RNAP III molecule involved in tRNA production in fast growing yeast cells has only 0.25-0.5 s to terminate as it approaches the terminator.…”

“…As noted, yeast 5S rRNA genes of 132 bp contains 2-3 elongating RNAP III molecules. 94 Many tRNA genes contain introns and are as long as this, and dimeric tRNA genes are significantly longer, apparently loaded with successive RNAP III.…”