Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast.
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-Delta6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Delta 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.
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 6A/Rm. B1A-13, Bethesda, MD 20892, USA.
Klaus H
KH
Nielsen
Leos
L
Valásek
Caroah
C
Sykes
Antonina
A
Jivotovskaya
Alan G
AG
Hinnebusch
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D039621:Eukaryotic Initiation Factor-3; C121939:HCR1 protein, S cerevisiae; D010448:Peptide Initiation Factors; D021122:Protein Subunits; C050998:Prt1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins