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Interaction of the RNP1 Motif in PRT1 with HCR1 Promotes 40S Binding of Eukaryotic Initiation Factor 3 in Yeast

Klaus H. Nielsen, Leos Valáek, Caroah Sykes, Antonina Jivotovskaya, and Alan G. Hinnebusch^*

Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Received 22 November 2005/ Returned for modification 23 December 2005/ Accepted 26 January 2006

We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts^– phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts^– phenotype is exacerbated by tif32-6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1 impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Centre for Structural Biology, Department of Molecular Biology, University of Aarhus, Science Park, 8000 Århus C, Denmark.

Present address: Laboratory of Regulation of Gene Expression, Division of Cellular and Molecular Microbiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

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