• Molecular Analysisof Saccharomyc escerevisiae RNA Polymerase I Core Factor Complex and its Interaction with Promoter DNA

      Knutson, Bruce; Jackobel, Ashleigh J (2020)
      Gene transcription and protein synthesis are essential molecular processes required for all living organisms. In eukaryotes, messages encoded within DNA are transcribed by three DNA-dependent RNA polymerases (Pols I-III) into ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), respectively. General transcription factors (GTFs) help recruit Pols to their appropriate gene promoters as well as facilitate template opening and transcription start site (TSS) selection. In Saccharomyces cerevisiae, the Pol I pre-initiation complex (PIC) is formed by numerous GTFs that include Upstream Activating Factor (UAF), Core Factor (CF), TATA-binding protein (TBP), and Rrn3. This unique set of GTFs engage ribosomal DNA (rDNA) through interactions with regulatory elements of the promoter known as the Upstream Activating Sequence (UAS) and the Core Element (CE). Here, we resolve the cryo-electron microscopy (cryo-EM) structure of CF bound to the rDNA promoter at 3.8Å near-atomic resolution and determine itsDNA binding preferences in which CF preferentially binds to the GC-minor groove. Briefly, our cryo-EM studies reveal that the CF-DNA interaction is mediated by two CF subunits, Rrn7 and Rrn11. We also found that the path of promoter DNA is relatively straight in the Pol I PIC, which is strikingly different from the bent promoters observed in structures of the Pols II/III PICs. We identified three states of CF engagement with promoter DNA (States 1-3) in which CF acts as a ratchet toforceDNA into the active site of the polymerase that facilitates the melting of the double-stranded DNA template in an ATP-independent manner, another unique feature of the Pol I system. Using in vitroDNA binding assays, we have identified a 12 base pair (bp) region within the CE that is necessary and sufficient for CF binding. We have also demonstrated that the human anticancer compound CX-5461 can inhibit yeast cell growth and blocks yeast CF binding to both yeast and human rDNA promoters in vitro. Furthermore, we show that the human Core Promoter Element (CPE) can functionally replace the yeast CE in a position-dependent manner. Together, these results reveal the unique molecular architecture of the Pol I PIC and suggest a conserved sequence-independent binding mechanism of CF with promoter DNA.
    • A trancriptomics based approach reveals the functional consequences of RNase MRP RNA mutations in yeast.

      Schmitt, Mark; Shafiuddin, Md (2018)
      RNase MRP is a eukaryotic ribonucleoprotein complex involved in multiple cellular functions that includes ribosomal RNA processing, primer generation for mitochondrial DNA replication and degradation of cell cycle related mRNAs. In Saccharomyces cerevisiae, the RNA component of RNase MRP is encoded by NME1. We have performed random deletion mutagenesis of RNase MRP RNA gene and isolated a mutation, nme1-91, that causes temperature sensitive growth defect on glycerol media. RNA analysis of nme1-91 showed that this mutant is mildly deficient in the 5.8S rRNA processing function of RNase MRP. Growth analysis and northern blotting of RNase MRP RNA mutations generated based on nme1-91 allele suggested that 3’-end nucleotide sequences of the nme1-91 allele contribute to its phenotype. Highcopy suppression screen identified tRNA modification gene NCS6 as a suppressor of nme1-91. Additionally, primary mode of suppression by NCS6 was found to be non-mitochondrial since NCS6 partially suppressed the nme1-91 phenotype on fermentable carbon source. Strains carrying a deletion of NCS6 in combination with nme1-91 showed a synthetic sick phenotype. Polysome profile analysis of nme1-91 revealed that 80S monosomal fraction accumulates in this mutant. Differential gene expression analysis of nme1-91 by RNAseq indicated that rRNA processing and cell cycle related genes become mis-regulated due to this mutation. A similar high-throughput sequencing based approach was also employed to investigate the transcriptional basis of positive genetic interactions between components of RNase MRP and nonsense-mediated decay pathway. A yeast strain bearing the nme1-P6 mutation in the RNA component of RNase MRP exhibits temperature-sensitive growth defect. This phenotype can be suppressed by deletion of NMD components. Differential gene expression analysis identified several mis-regulated biological processes in nme1-P6 and Δupf1 strains. Comparative transcriptomic analysis suggested that suppression of nme1-P6 phenotype by Δupf1 is accompanied by large shift in gene expression pattern towards Δupf1 strain. Moreover, the majority of direct targets of NMD were not down-regulated in nme1-P6 indicating that the effect of NMD on nme1-P6 might be due to increased degradation on mRNAs that are not targeted by NMD in normal conditions. Taken together, these results show that mutations of RNase MRP RNA can modulate diverse biological processes.