1. A yeast prion provides a mechanism for genetic variation and phenotypic diversity Heather L. True & Susan L. Lindquist Department of Molecular Genetics and Cell Biology Howard Hughes Medical Institute University of Chicago Nature – 28 September 2000

2. A biological problem Individual spontaneous genetic changes may be deleterious Happy, living cells Dead cells

3. A biological problem Individual spontaneous genetic changes may be deleterious But what if several independent changes could occur, that would work together to produce a beneficial new phenotype? Happy, living cells Dead cells ++

4. What if there is a way to accumulate genetic changes “behind closed doors” and then allow them to express together to produce a new form or functions?

6. A proposed mechanism Gene duplication is a common event A duplicated gene that is retained in an inactive state could accumulate variations Inactive genes reactivated all at once; cell benefits from combinatorial changes HOW?

7. STOP RNA transcript polypeptide ribosome Sup45Sup35 Sup35 = yeast translation termination factor forms complex with Sup45 Sup45 recognizes STOP codon Sup35 facilitates release of nascent polypeptide

8. Sup35 ( [psi-] ) Has a prion form [PSI+] [PSI+] reduces fidelity of translation termination → causes ribosomes to read through stop codons → suppresses nonsense mutations produces heritable changes in phenotype with no changes in nucleic acids

9. NMC N-terminal region Middle region C-terminal region Sup35 has three distinct regions C-terminal region: responsible for translation termination; essential for viability N-terminal & Middle regions: allow Sup35 to acquire stable prion conformation allow cell to switch between [PSI+] and [psi-] states can be deleted in [psi-] cells with no apparent effect deletion results in loss of prion, restoration of translation termination fidelity

10. Diverse growth phenotypes produced by [PSI+] Compared growth characteristics of [PSI+] and [psi-] cells in >150 phenotypic assays: fermentable & non-fermentable carbon sources simple & complex nitrogen sources in presence of salts & metals with inhibitors of diverse cellular processes: –DNA replication –Signalling –Protein glycosylation –Microtubule dynamics general stress conditions different temperatures

11. Diverse growth phenotypes produced by [PSI+] Examined cells from seven different genetic backgrounds to distinguish traits acquired from [PSI+] state from traits arising in specific genomes Strains 74-D694 33G-D373 SL1010-1A D1142-1A 5V-H19 10B-H49u BSC783/4c used guanidine hydrochloride curing to create stable, isogenic pairs ([PSI+] & [psi-] phenotypic differences would be due to [PSI+] effects on gene expression, not genetic changes from long-term culture each strain had a different intrinsic level of [PSI+]-mediated nonsense suppression low- to high- levels of [PSI+] nonsense suppression activity represented in selection of strains

12. Diverse growth phenotypes produced by [PSI+] CONTROL: Examined NM deletion strains in two genetic backgrounds To detect phenotypes that may be due to loss of NM region function (which may occur with prion formation due to occlusion), in the absence of [PSI+] activity Strains 74-D694  NM 33G-D373  NM

13. Carefully controlled experimental procedures Strain pairs examined at same growth stage –Mid-log phase cells grown in rich medium (YPD) –Serially diluted 5X, spotted onto test plates Tested amino acid supplementation in parallel experiments –No changes in growth patterns phenotypic differences were not owing to effects of auxotrophic markers the strains contain –Most strains carried adenine mutations, which turn red in the absence of adenine ([PSI+] suppressed these mutations some of the time) YPD control plates were periodically interspersed with test plates –Assured consistent spotting of cells, with same densities Performed repeat experiments –Same phenotypic variances were observed each time

14. Marked growth differences observed between isogenic [PSI+] & [psi-] derivatives on many test plates Change in colony morphology Enhanced growth in [psi-] derivative Enhanced growth in [PSI+] derivative * * Stronger color = stronger effect * Growth scored in two ways: 1) numbers designate the highest dilution that produced significant growth in repeat experiments 2) letters indicate growth rates: R = rapid; M = medium; S = slow V = very slow; NG = no growth Change in growth in corresponding DNM strain Indicated by superscript: a = growth similar to [psi-] b = growth similar to [PSI+] c = different from both [psi-] & [PSI+] For stress assays: Growth rates: H = high; M = medium; L = low + = slightly increased tolerance

15. Change in colony morphology Enhanced growth in [psi-] derivative Enhanced growth in [PSI+] derivative * * Stronger color = stronger effect * Growth scored in two ways: 1) numbers designate the highest dilution that produced significant growth in repeat experiments 2) letters indicate growth rates: R = rapid; M = medium; S = slow V = very slow; NG = no growth Change in growth in corresponding DNM strain Indicated by superscript: a = growth similar to [psi-] b = growth similar to [PSI+] c = different from both [psi-] & [PSI+] For stress assays: Growth rates: H = high; M = medium; L = low + = slightly increased tolerance

16. [PSI+] does not alter growth on YPD at pH 6.8 [PSI+] causes strain-specific changes at pH 6.0

17. [PSI+] does not alter growth on YPD at pH 6.8 [PSI+] causes strain-specific changes at pH 6.0 [psi-] grew better here [PSI+] grew better here

18. [PSI+] affects growth in different ways in different genetic backgrounds

19. Colony morphology changes Cells grown in YPD, spotted onto medium with potassium acetate as sole carbon source [PSI+] colonies [psi-] coloniesSpontaneous appearance of [PSI+] colony in a group of [psi-] cells Cells can switch from [PSI+] to [psi-], and vice versa

20. Stress tolerance Cells spotted on plates with ethanol gradient 0 10% Cells in log phase were incubated at 37ºC for 30 min. then transferred to 50ºC for the times indicated

21. [PSI+] negatively affected growth of all strain backgrounds when cells were plated with 5mM ZnCl 2

22. Divergent range of phenotypes Strong enhancement, but only at high concentration of metal Both these strains affected similarly by [PSI+] Same strains affected very differently by [PSI+] [PSI+] has little effect on most strains, but strongly affects one [PSI+] enhances growth on one strain, inhibits on others, under same conditions

23. Divergent range of phenotypes in nearly half the conditions tested, [PSI+] had a substantial impact in growth of at least some strains in > 25% of tests, the impact on the strain was beneficial each strain exhibited a unique combination of phenotypes in response to [PSI+]

24. Are there common biological effects of [PSI+] ? Enhance stress tolerance? no significant difference between [PSI+] &[psi-] in either ethanol tolerance or thermotolerance conditions Affect all strains in the same way? only once: ZnCl 2 affected all six strains capable of growth in presence of calcofluor white Possible biological effect of [PSI+] on zinc metabolism or cell wall biosynthesis

25. NM – specific phenotypes? [PSI+] had both enhancing and inhibiting effects on growth in the presence of benomyl no common biological effect DNM mutants grew better in the presence of benomyl than either [PSI+] or [psi-] variants DNM phenotypes were mostly different from both [PSI+] & [psi-] The NM region of Sup35 may play a role in yeast biology in addition to its role in [PSI+] formation

26. CONCLUSIONS: [PSI+] element of S. cerevisiae provides a means to activate silent genetic information to produce new heritable phenotypes as a suppressor of nonsense mutations, [PSI+] provides mechanism for read- through of naturally-occuring STOP codons, providing potential access to genetic variability in duplicate genes or in 3’ non-coding regions allows alternative heritable phenotypes to be encoded by a single genome provides possibility of survival in fluctuating environment provides a means for evolution of new traits Yeast cells spontaneously switch between [PSI+] & [psi-] states both phenotypes are available, for adaptation to changing environment