The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively.

Spt5p is a universally conserved transcription factor that plays multiple roles in eukaryotic transcription elongation. Spt5p forms a heterodimer with Spt4p and collaborates with other transcription factors to pause or promote RNA polymerase II transcription elongation. We have shown previously that Spt4p and Spt5p also influence synthesis of ribosomal RNA by RNA polymerase (Pol) I; however, previous studies only characterized defects in Pol I transcription induced by deletion of SPT4. Here we describe two new, partially active mutations in SPT5 and use these mutant strains to characterize the effect of Spt5p on Pol I transcription. Genetic interactions between spt5 and rpa49Δ mutations together with measurements of ribosomal RNA synthesis rates, rDNA copy number, and Pol I occupancy of the rDNA demonstrate that Spt5p plays both positive and negative roles in transcription by Pol I. Electron microscopic analysis of mutant and WT strains confirms these observations and supports the model that Spt4/5 may contribute to pausing of RNA polymerase I early during transcription elongation but promotes transcription elongation downstream of the pause(s). These findings bolster the model that Spt5p and related homologues serve diverse critical roles in the control of transcription.

Radon exposure is rising steadily within the modern North American residential environment, and is increasingly uniform across seasons.

Human-made buildings can artificially concentrate radioactive radon gas of geologic origin, exposing occupants to harmful alpha particle radiation emissions that damage DNA and increase lung cancer risk. We examined how North American residential radon exposure varies by modern environmental design, occupant behaviour and season. 11,727 residential buildings were radon-tested using multiple approaches coupled to geologic, geographic, architectural, seasonal and behavioural data with quality controls. Regional residences contained 108 Bq/m3 geometric mean radon (min < 15 Bq/m3; max 7,199 Bq/m3), with 17.8% ≥ 200 Bq/m3. Pairwise analysis reveals that short term radon tests, despite wide usage, display limited value for establishing dosimetry, with precision being strongly influenced by time of year. Regression analyses indicates that the modern North American Prairie residential environment displays exceptionally high and worsening radon exposure, with more recent construction year, greater square footage, fewer storeys, greater ceiling height, and reduced window opening behaviour all associated with increased radon. Remarkably, multiple test approaches reveal minimal winter-to-summer radon variation in almost half of properties, with the remainder having either higher winter or higher summer radon. This challenges the utility of seasonal correction values for establishing dosimetry in risk estimations, and suggests that radon-attributable cancers are being underestimated.

Transcription elongation by RNA polymerase I is linked to efficient rRNA processing and ribosome assembly.

The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant shows significant defects in rRNA processing and ribosome assembly. Taken together, these data suggest that transcription of rRNA by RNA polymerase I is linked to rRNA processing and maturation. Thus, RNA polymerase I, elongation factors, and rRNA sequence elements appear to function together to optimize transcription elongation, coordinating cotranscriptional interactions of many factors/snoRNAs with pre-rRNA for correct rRNA processing and ribosome assembly.

Two reactions of Haloferax volcanii RNA splicing enzymes: joining of exons and circularization of introns.

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon-intron junctions, producing 2',3'-cyclic phosphate and 5'-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3' terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5' and 3' flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3',5'-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.