Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

Abstract

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...

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

“…Although the tertiary structure of 5S rRNA has been determined for the free molecule (Funari et al 2000;Lorenz et al 2000) and its isolated domains (Betzel et al 1994;Xiong and Sundaralingam 2000;Huber et al 2001), and as a part of a ribosomal complex (Ban et al 2000;Harms et al 2001;Spahn et al 2001;Yusupov et al 2001), its precise function in protein synthesis is not fully understood. Biochemical studies with ribosomes from Escherichia coli led to the hypothesis that 5S rRNA acts to facilitate communication between the different functional centers, helping to coordinate the multiple events catalyzed by the ribosome (Bogdanov et al 1995;Dokudovskaya et al 1996), and this view was further supported by a later study of yeast ribosomes (Smith et al 2001). There are 100-200 rDNA genes in the haploid yeast genome, all of which are located in a single tandem array on chromosome XII called the RDN1 locus (Petes 1979a, b).…”

“…Subsequently, 10 4 CFU (5 μl) aliquots, and tenfold dilutions from the same cultures thereof were spotted either onto rich medium and incubated at 15, 30 and 37°C, or incubated at 30°C on rich medium containing anisomycin (10 μg/ml) or sparsomycin (30 μg/ml). Assays for programmed ribosomal frameshifting followed previously described protocols (Smith et al 2001;Harger and Dinman 2003). These involve the use of 0-frame control and −1 or +1 ribosomal frameshift test vectors in which the production of a reporter enzyme (either firefly luciferase of β-galactosidase) is dependent upon a programmed ribosomal frameshift event.…”

“…In a previous study, we described the generation of a near saturation library of 5S rRNA variants; specifically, 246 out of the 363 possible alleles of the yeast molecule were expressed from a high-copy-number 2μ vector (Smith et al 2001). With the aim of examining the effects of the 5S rRNA mutants in the absence of the wild-type molecule, we obtained the strain NOY1049, in which the chromosomal 5S rDNA repeats were completely deleted (Wai et al 2000).…”

“…LEU [LA-HN M 1 ]), and of strains harboring mutant alleles of 5S rDNA on a 2μ-TRP1 vector was described previously (Smith et al 2001). The starting strain was based on NOY891 (Oakes et al 1998), which was subsequently found to contain four telomere-proximal 5S rDNA repeats on chromosome XII (Wai et al 2000).…”

Ribosomal Protein L3: Influence on Ribosome Structure and Function

“…The chain formed by these helices of the large subunit rRNA makes a connection between the peptidyltransferase center, the GTPase-associated center of the large subunit, and 5S rRNA. These interconnections support an allosteric model in which the various ribosomal functions are coordinated by the intramolecular crosstalk between all of the different functional centers of this complex macromolecule (32). Mid-log phase cultures of isogenic strains harboring the various plasmid-borne RPL3 alleles were spotted in 10-fold dilutions onto SD-trp alone (No Drug), SD-trp containing sparsomycin (35μg/ml), or SD-trp plus anisomycin (25μg/ml).…”