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Molecular and Cellular Biology, July 2005, p. 5480-5491, Vol. 25, No. 13
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.13.5480-5491.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation

Chingakham Ranjit Singh,1,{dagger} Cynthia Curtis,2,{dagger} Yasufumi Yamamoto,1,{dagger} Nathan S. Hall,1 Dustin S. Kruse,1 Hui He,1 Ernest M. Hannig,2* and Katsura Asano1*

Program for Molecular Cellular and Developmental Biology, Division of Biology, Kansas State University, Manhattan, Kansas 66506,1 Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 750832

Received 7 January 2005/ Returned for modification 25 February 2005/ Accepted 8 April 2005

The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts– phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd– phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAiMet ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn– phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.

* Corresponding author. Mailing address for Katsura Asano: Division of Biology, 258 Chalmers Hall, Kansas State University, Manhattan, KS 66506. Phone: (785) 532-0116. Fax: (785) 532-6653. E-mail: kasano{at}ksu.edu. E-mail for Ernest M. Hannig: hannig{at}utdallas.edu.

{dagger} These authors contributed equally to this work.

Molecular and Cellular Biology, July 2005, p. 5480-5491, Vol. 25, No. 13
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.13.5480-5491.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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