Growth phenotypes of “pure” 5S rRNA mutants. Mid-logarithmically growing JD1253 cells were diluted to 2 × 10⁷ colony forming units (CFU)/ml. Subsequently, aliquots (5 μl) containing 10⁴ CFU, and tenfold dilutions from the same cultures thereof were spotted either onto rich medium (YPAD) and incubated at 15, 30, and 37°C, or onto YPAD containing anisomycin (10 μg/ml) or sparsomycin (30 μg/ml) and incubated at 30°C

Chemical protection analyses. Ribosomes purified from JD1253 cells expressing either wild-type or mutant 5S rRNAs were chemically probed with DMS, kethoxal and CMCT. The products of reverse transcriptase primer extension reactions were subjected to PAGE analysis on denaturing PA-urea gels, and visualized by autoradiography. Left panel Representative autoradiographs showing altered reactivities of bases in 5S rRNA (top and middle), and 25S rRNA (bottom). Right panel Summary of the effects of pure mutants mapped onto 5S rRNA and the helix 95-sarcin/ricin loop (SRL) region of 25S rRNA. The open circles indicate positions of wild-type bases, and the arrows indicate the relevant mutant alleles. The color-coded bars indicate which mutant 5S rRNAs alter the chemical reactivities of other similarly coded, circled, bases in 5S and 25S rRNAs

a, b Semi dominant effects of 5S rRNA alleles on programmed ribosomal frameshifting and virus propagation. a Effects on −1 PRF and killer virus propagation. b Effects on +1 PRF and Ty1 retrotransposition. JD1111 cells expressing wild-type or mixtures of wild-type and mutant 5S rRNA alleles were transformed with monocistronic lacZ-based 0-frame, −1 or +1 frameshift reporter plasmids and L–A virus promoted −1, or Ty1 promoted +1 PRF efficiencies were determined as previously described (Dinman et al. 1991; Dinman and Wickner 1992). Alleles of 5S rRNA are shown on the X-axis and PRF efficiencies are indicated on the Y-axis. The error bars correspond to standard error. In panel a, the different shadings indicate inhibitory effects of 5S rRNA alleles on maintenance of either the M₁ satellite virus alone or both L– A and M₁, as previously described (Smith et al. 2001). In panel b, the shadings correspond to inhibitory effects of 5S rRNA alleles on Ty1 retrotransposition frequencies assessed using pJEF1105

a–c Correlation of 5S rRNA structure with function. Mutations in 5S rRNA that produced phenotypic and physical changes are mapped onto the high-resolution structure of the yeast ribosome described by Spahn et al. (2004). a Mapping of sites affected in “pure” mutants onto 5S and 25S rRNA structures. Positions marked in green in 5S rRNA show locations of A20, A79, and U81. Positions marked in red show the locations of bases whose chemical reactivities were altered as a consequence of the 5S rRNA mutations. b, c “Mixed” 5S rRNA alleles that affected +1 (b) or −1 PRF (c). Mutations at bases shown in red stimulated PRF, and those shown in blue inhibited PRF

Direct rRNA sequence analyses of “pure” 5S rRNA mutants. rRNAs were extracted from ribosomes purified from JD1253 cells expressing wild-type or mutant forms of 5S rRNA. Oligonucleotides complementary to 5S rRNA were labeled with γ[³²P]ATP using T4 polynucleotide kinase, and annealed with 5S rRNA isolated from purified ribosomes. Primer-extension reactions were performed using AMV reverse transcriptase, fractionated by electrophoresis through denaturing 12% polyacrylamide-urea gels, and labeled bands were visualized by autoradiography

a–d Effects of pure 5S rRNA mutants on programmed ribosomal frameshifting and virus propagation. JD1253 cells expressing the indicated “pure” 5S rRNA were assayed with respect to the following phenotypes. a Cells were transformed with 0-frame control and L–A derived −1 PRF test dual luciferase reporter plasmids, and −1 PRF efficiencies were determined as described by Harger and Dinman (2003). All assays were replicated at least 30 times, and standard errors (represented by the error bars) were calculated as described by Jacobs and Dinman (2004). b Programmed +1 ribosomal frameshifting was analyzed using a Ty1 derived +1 PRF signal cloned into the dual luciferase reporter plasmids as described above. c Total nucleic acids were extracted from JD1253 cells expressing the indicated 5S rRNA alleles, fractionated by electrophoresis on a 1.5% native agarose gel, and stained with ethidium bromide. Genomic DNA (gDNA), viral dsRNAs, and rRNAs are indicated. L-BC is a dsRNA virus unrelated to L–A and M₁. d Cells were transformed with pJEF1105, a galactose-inducible Ty1 cDNA clone containing the neo^r selectable reporter, incubated at 20°C for 4 days on medium containing 2% galactose, replica plated onto medium containing 100 μg/ml 5-FOA, and incubated at 30°C to select for cells that had lost pJDF1105. Viable colonies were grown on YPAD medium overnight, and 10-fold dilutions of cells ranging from 10⁶ to 10² CFU were spotted onto YPAD medium containing 100 μg/ml of Genticin. In parallel, 10⁵–10¹ CFU were spotted onto YPAD medium alone

Direct rRNA sequence analyses of “mixed” 5S rRNA mutants. rRNAs were extracted from ribosomes purified from JD1111 cells expressing wild-type or mixtures of mutant and wild-type forms of 5S rRNA. Oligonucleotides complementary to 5S rRNA were labeled with γ[³²P]ATP using T4 polynucleotide kinase, and annealed with 5S rRNA isolated from purified ribosomes. Primer-extension reactions were performed using AMV reverse transcriptase, fractionated by electrophoresis through denaturing 12% polyacrylamide-urea gels, and visualized by autoradiography. The asterisks indicate the 5S rRNA mutations in the mixed populations

a, b Allelic variants of 5S rRNAs in eukaryotic genomes. a Conservation of multiple 5S rDNA alleles in eukaryotic genomes. GenBank was first queried for 5S rRNA sequences according to species, and the resulting sequences were used in BLAST searches (Altschul et al. 1990) to identify homologous sequences in the database. Sequences were hand curated to ensure their validity, and then aligned with one another as described in Materials and methods. RDN5-som and RDN5-ooc show the sequences of the hybrid yeast/Xenopus clones. Color coding is used to denote base substitutions. b Semi-dominant effects of naturally occurring yeast and hybrid yeast/Xenopus 5S rRNA alleles on L–A directed −1, and Ty1 promoted +1 PRF. Using the yeast RDN5-1 allele cloned into a high-copy-number 2μ vector, oligonucleotide-primed site-directed mutagenesis was used to create the other six naturally occurring yeast RDN5 alleles (RDN5-2 to RDN5-7), and the hybrid yeast/Xenopus RDN5-ooc and RDN5-som alleles (see Fig. 6a). These alleles were episomally expressed in JD932 cells, a wild-type yeast strain containing a full complement of chromosomal rDNA genes. Programmed −1 and +1 ribosomal frameshifting efficiencies were monitored as described in Fig. 6