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Molecular and Cellular Biology, December 2005, p. 10863-10874, Vol. 25, No. 24
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.24.10863-10874.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Functionally Important Amino Acids of Ribosomal Protein L3 by Saturation Mutagenesis

Arturas Meskauskas, Alexey N. Petrov, and Jonathan D. Dinman*

Department of Cell Biology and Molecular Genetics, Microbiology Building Room 2135, University of Maryland, College Park, Maryland 20742

Received 2 June 2005/ Returned for modification 5 July 2005/ Accepted 27 September 2005

There is accumulating evidence that many ribosomal proteins are involved in shaping rRNA into their functionally correct conformations through RNA-protein interactions. Moreover, although rRNA seems to play the central role in all aspects of ribosome function, ribosomal proteins may be involved in facilitating communication between different functional regions in ribosome, as well as between the ribosome and cellular factors. In an effort to more fully understand how ribosomal proteins may influence ribosome function, we undertook large-scale mutational analysis of ribosomal protein L3, a core protein of the large subunit that has been implicated in numerous ribosome-associated functions in the past. A total of 98 different rpl3 alleles were genetically characterized with regard to their effects on killer virus maintenance, programmed –1 ribosomal frameshifting, resistance/hypersensitivity to the translational inhibitor anisomycin and, in specific cases, the ability to enhance translation of a reporter mRNA lacking the 5' 7mGppp cap structure and 3' poly(A) tail. Biochemical studies reveal a correlation between an increased affinity for aminoacyl-tRNA and the extent of anisomycin resistance and a decreased peptidyltransferase activity and increased frameshifting efficiency. Immunoblot analyses reveal that the superkiller phenotype is not due to a defect in the ability of ribosomes to recruit the Ski-complex, suggesting that the defect lies in a reduced ability of mutant ribosomes to distinguish between cap+/poly(A)+ and cap–/poly(A)– mRNAs. The results of these analyses are discussed with regard to how protein-rRNA interactions may affect ribosome function.

* Corresponding author. Mailing address: Department of Cell Biology and Molecular Genetics, Microbiology Building Room 2135, University of Maryland, College Park, MD 20742. Phone: (301) 405-0981. Fax: (301) 314-9489. E-mail: dinman{at}umd.edu.

Molecular and Cellular Biology, December 2005, p. 10863-10874, Vol. 25, No. 24
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.24.10863-10874.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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