RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5 ′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3′ -extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3 ′ -extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5 ′ -exonuclease Rat1.[Supplemental material is available for this article.]Transcription of nuclear DNA in eukaryotes is carried out by at least three different RNA polymerases. RNA polymerase III (RNAPIII) is specialized for high-level synthesis of small noncoding RNAs. The most abundant products of RNAPIII-dependent transcription are the 5S rRNA and the many pre-tRNA species. In addition, RNAPIII synthesizes numerous, less abundant small RNAs that are involved in diverse cellular processes, including protein translocation and the processing of pre-rRNA and pre-tRNAs (Dieci et al. 2013). The nuclear genome of Saccharomyces cerevisiae contains 275 actively transcribed tRNA genes (including a tRNA of undetermined specificity [tX(XXX)D]). These are grouped into 20 isotypes, each charged with a single amino acid, which are subdivided into 41 isoacceptors that each recognize the same anticodon sequence(s) (Hani and Feldmann 1998; Chan and Lowe 2009). The reported lengths of the primary transcripts vary between 72 and 133 nucleotides (nt), and ∼25% of pre-tRNAs include introns. Primary pre-tRNA transcripts undergo 5 ′ and 3 ′ maturation and intron excision to generate the mature tRNAs. In tRNA genes, the transcription machinery recognizes conserved promoter elements, termed box A and box B, which are located within the transcribed region and form a bipartite binding site for the six-subunit basal transcription factor TFIIIC (Acker et al. 2013). Box A starts at position +8 of the mature tRNA, and the transcription start site is most frequently located 18-20 nt upstream (Dieci et al. 2013). Within yeast tRNA genes, boxes A and B are localized 31-93 nt apart and correspond to the universally conserved D-and T-loops in the tRNA structure. Internally located A and B boxes are...

RNA polymerase III (Pol III) transcribes a limited set of short genes in eukaryotes producing abundant small RNAs, mostly tRNA. The originally defined yeast Pol III transcriptome appears to be expanding owing to the application of new methods. Also, several factors required for assembly and nuclear import of Pol III complex have been identified recently. Models of Pol III based on cryo-electron microscopy reconstructions of distinct Pol III conformations reveal unique features distinguishing Pol III from other polymerases. Novel concepts concerning Pol III functioning involve recruitment of general Pol III-specific transcription factors and distinctive mechanisms of transcription initiation, elongation and termination. Despite the short length of Pol III transcription units, mapping of transcriptionally active Pol III with nucleotide resolution has revealed strikingly uneven polymerase distribution along all genes. This may be related, at least in part, to the transcription factors bound at the internal promoter regions. Pol III uses also a specific negative regulator, Maf1, which binds to polymerase under stress conditions; however, a subset of Pol III genes is not controlled by Maf1. Among other RNA polymerases, Pol III machinery represents unique features related to a short transcript length and high transcription efficiency.

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Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in de novo synthesis of guanidine nucleotides.

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.