The eukaryotic initiation factor (eIF) 4G HEAT domain promotes translation re-initiation in yeast both dependent on and independent of eIF4A mRNA helicase.
Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.
Watanabe
Ryosuke
R
Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.
Murai
Marcelo Jun
MJ
Singh
Chingakham Ranjit
CR
Fox
Stephanie
S
Ii
Miki
M
Asano
Katsura
K
D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D018387:Codon, Initiator / Q000235:genetics; D039601:Eukaryotic Initiation Factor-4A / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D010442:Peptide Chain Initiation, Translational; D010455:Peptides; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D050976:Basic-Leucine Zipper Transcription Factors; D018387:Codon, Initiator; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; C501391:GCN4 protein, S cerevisiae; D010455:Peptides; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D039601:Eukaryotic Initiation Factor-4A
Science. 1997 Mar 7;275(5305):1468-71
9045610
Cell. 2002 Dec 27;111(7):1015-25
12507428
J Biol Chem. 1995 Mar 17;270(11):5764-71
7890705
EMBO J. 2006 Oct 4;25(19):4537-46
16990799
Methods Enzymol. 2007;429:139-61
17913622
Mol Microbiol. 2004 Feb;51(4):987-1001
14763975
Cell. 2003 Dec 12;115(6):739-50
14675538
J Biol Chem. 1997 Oct 24;272(43):27042-52
9341143
Proc Natl Acad Sci U S A. 2005 Nov 8;102(45):16164-9
16254050
Genes Dev. 2004 Jan 1;18(1):62-75
14701882
Genes Dev. 2003 Nov 15;17(22):2786-97
14600024
Nat Rev Mol Cell Biol. 2010 Feb;11(2):113-27
20094052
Science. 2005 Dec 2;310(5753):1513-5
16322461
EMBO J. 2003 Apr 15;22(8):1909-21
12682023
Mol Cell. 2005 Jan 21;17(2):265-75
15664195
Mol Cell Biol. 1999 Jan;19(1):173-81
9858542
Genes Dev. 2005 Sep 15;19(18):2212-23
16166382
Cell. 2008 Dec 26;135(7):1237-50
19109895
Mol Cell Biol. 1991 Jan;11(1):486-96
1986242
Mol Cell. 2007 Apr 13;26(1):41-50
17434125
Genes Dev. 1997 Sep 15;11(18):2396-413
9308967
EMBO J. 2008 Jun 4;27(11):1609-21
18464793
Cell. 2005 Jan 14;120(1):49-58
15652481
Proc Natl Acad Sci U S A. 1997 May 13;94(10):5201-6
9144215
Cell. 1986 Apr 25;45(2):201-7
3516411
Mol Cell. 2005 Oct 28;20(2):251-62
16246727
Mol Cell Biol. 1997 Dec;17(12):6876-86
9372919
Mol Cell Biol. 1999 Aug;19(8):5557-64
10409745
Mol Cell Biol. 1994 Apr;14(4):2616-28
8139562
Annu Rev Microbiol. 2005;59:407-50
16153175
Mol Cell Biol. 1988 Jul;8(7):2955-63
3043200
EMBO J. 2001 May 1;20(9):2326-37
11331597
Genes Dev. 2008 Sep 1;22(17):2414-25
18765792
Genes Dev. 2007 Jun 1;21(11):1280-7
17545463
J Biomol NMR. 2001 May;20(1):11-4
11430750
Mol Cell. 2001 Jan;7(1):193-203
11172724
Mol Cell Biol. 2003 Aug;23(15):5431-45
12861028
J Biol Chem. 2008 Aug 8;283(32):22063-75
18502752
Biochemistry. 2005 Sep 20;44(37):12265-72
16156639
RNA. 2003 Jul;9(7):871-80
12810920
Genes Dev. 2007 May 15;21(10):1217-30
17504939
J Biol Chem. 1977 Nov 10;252(21):7842-9
914843
Cell. 2009 Feb 6;136(3):447-60
19203580