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Molecular and Cellular Biology, December 2001, p. 8264-8275, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8264-8275.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

Maria W. Smith,1,dagger Arturas Meskauskas,1 Pinger Wang,1 Petr V. Sergiev,2 and Jonathan D. Dinman1,3,4,5,*

Department of Molecular Genetics and Microbiology,1 Graduate Program in Molecular Biosciences,3 and Program in Computational Molecular Biology Graduate Studies,5 Rutgers University and University of Medicine and Dentistry of New Jersey, and Cancer Institute of New Jersey,4 Piscataway, New Jersey 08854, and Department of Chemistry, Moscow Lomonosov State University, Moscow 119899, Russia2

Received 3 July 2001/Returned for modification 2 August 2001/Accepted 17 September 2001

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.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Program in Computational Molecular Biology Graduate Studies at Rutgers University and UMDNJ, 675 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-4670. Fax: (732) 235-5223. E-mail: dinmanjd{at}umdnj.edu.

dagger Present address: Department of Microbiology, University of Washington Regional Primate Research Center, Seattle, WA 98195.

Molecular and Cellular Biology, December 2001, p. 8264-8275, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8264-8275.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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