Translation includes initiation, elongation, and termination, followed by ribosome recycling. We characterize a new sequence element in 5' untranslated regions that consists of an adjacent start and stop codon and thereby excludes elongation. In these start-stop elements, an initiating ribosome is simultaneously positioned for termination without having translocated. At the example of activating transcription factor 4 (ATF4), we demonstrate that start-stops modify downstream re-initiation, thereby repressing translation of upstream open reading frames and enhancing ATF4 inducibility under stress. Start-stop elements are abundant in both mammals and yeast and affect key regulators such as DROSHA and the oncogenic transcription factor NFIA. They provide a unique regulatory layer that impedes ribosome scanning without the energy-expensive peptide production that accompanies upstream open reading frames.

Ribosome biogenesis in eukaryotes is supported by hundreds of ribosomal RNA (rRNA) gene copies that are encoded in the ribosomal DNA (rDNA). The multiple copies of rRNA genes are thought to have low sequence diversity within one species. Here, we present species-wide rDNA sequence analysis in Saccharomyces cerevisiae that challenges this view. We show that rDNA copies in this yeast are heterogeneous, both among and within isolates, and that many variants avoided fixation or elimination over evolutionary time. The sequence diversity landscape across the rDNA shows clear functional stratification, suggesting different copy-number thresholds for selection that contribute to rDNA diversity. Notably, nucleotide variants in the most conserved rDNA regions are sufficiently deleterious to exhibit signatures of purifying selection even when present in only a small fraction of rRNA gene copies. Our results portray a complex evolutionary landscape that shapes rDNA sequence diversity within a single species and reveal unexpectedly strong purifying selection of multi-copy genes.

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The ribosomal DNA (rDNA) is a conserved but highly unstable multi-gene locus that codes for the RNA components of the ribosome (rRNA). Despite being essential for protein translation and survival, rRNA gene-copy numbers fluctuate frequently. Here, we describe a novel rDNA intermediate that may be involved in these copy-number changes in Saccharomyces cerevisiae. Two-dimensional gel analyses revealed a structure that includes a large single-stranded tail and arises from the promoter of the 35S rRNA gene. Formation of this intermediate is unaffected by reduced 35S transcription but relies on topoisomerase I (Top1). Unexpectedly, intermediate formation is also independent of S phase or the rDNA-instability factor Fob1, even though Fob1 is mediates the major peak of Top1 cleavage complexes in the rDNA. Indeed, we find that the known rDNA instability phenotypes of top1 mutants, including increased formation of extrachromosomal rDNA circles, elevated genetic marker loss, and instability of critically short rDNA arrays, are also largely independent of Fob1. We therefore speculate that failure to form this intermediate leads to rDNA instability in top1 mutants.