The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons.
The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.
Chiu
Wen-Ling
WL
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.
Wagner
Susan
S
Herrmannová
Anna
A
Burela
Laxminarayana
L
Zhang
Fan
F
Saini
Adesh K
AK
Valásek
Leos
L
Hinnebusch
Alan G
AG
D000595:Amino Acid Sequence; D000818:Animals; D018387:Codon, Initiator / Q000235:genetics / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010448:Peptide Initiation Factors; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018387:Codon, Initiator; D039621:Eukaryotic Initiation Factor-3; C121939:HCR1 protein, S cerevisiae; D010448:Peptide Initiation Factors; D021122:Protein Subunits; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins