rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome.The ribosome is the central component of an extremely accurate cellular protein synthesis apparatus. Its function is to efficiently and accurately decode mRNAs. Eukaryotic ribosomes contain four rRNAs: three large-subunit-associated rRNAs (28S-25S in eukaryotes and 23S in prokaryotes, plus 5.8S and 5S) and the small-subunit rRNA (18S in eukaryotes and 16S in prokaryotes). Although these rRNAs were initially thought to provide the scaffolding for the enzymatic ribosomal proteins, early reconstitution and depletion experiments hinted at broader roles for these molecules (reviewed in references 37 and 39), and it is now clear that the rRNAs are the central players in the reactions catalyzed by ribosomes and that the individual rRNAs are actively involved in different ribosomal functions (reviewed in references 9, 30, 38, 41, and 63). Thus, understanding the molecular basis of rRNA structure and function is central to furthering our comprehension of the translational apparatus.The 5S rRNA is a component of the large ribosomal subunit in all living organisms (with the exception of mitochondrial ribosomes) (see reference 27 for a review). In eukaryotic cells, 5S rRNA is synthesized in the nucleolus by RNA polymerase III, processed into its mature form, and then imported into the nucleus, where it associates with ribosomal protein L5. The 5S-L5 ribonuclear particle is reimported into the nucleolus, where it is assembled into the ce...

Nonsense-mediated mRNA decay (NMD) directs rapid degradation of premature termination codon (PTC)-containing mRNAs, e.g. those containing frameshift mutations. Many viral mRNAs encode polycistronic messages where programmed -1 ribosomal frameshift (-1 PRF) signals direct ribosomes to synthesize polyproteins. A previous study, which identified consensus -1 PRF signals in the yeast genome, found that, in contrast to viruses, the majority of predicted -1 PRF events would direct translating ribosomes to PTCs. Here we tested the hypothesis that a -1 PRF signal can function as a cis-acting mRNA destabilizing element by inserting an L-A viral -1 PRF signal into a PGK1 reporter construct in the 'genomic' orientation. The results show that even low levels of -1 PRF are sufficient to target the reporter mRNA for degradation via the NMD pathway, with half-lives similar to messages containing in-frame PTCs. The demonstration of an inverse correlation between frameshift efficiency and mRNA half-lives suggests that modulation of -1 PRF frequencies can be used to post-transcriptionally regulate gene expression. Analysis of the mRNA decay profiles of the frameshift-signal- containing reporter mRNAs also supports the notion that NMD remains active on mRNAs beyond the 'pioneer round' of translation in yeast.