Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

Abstract In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the −3 nucleotide of the ‘Kozak’ context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui− cells (Ssu− phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui− phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd− phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

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Prediction of translation initiation sites in human mRNA sequences with AUG start codon in weak Kozak context: A neural network approach

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2008.03.008 ◽

2008 ◽

Vol 369 (4) ◽

pp. 1166-1168 ◽

Cited By ~ 10

Author(s):

Suhas Tikole ◽

Ramasubbu Sankararamakrishnan

Keyword(s):

Neural Network ◽

Translation Initiation ◽

Start Codon ◽

Network Approach ◽

Neural Network Approach ◽

Kozak Context

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uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

Genetics ◽

10.1093/genetics/iyab176 ◽

2021 ◽

Author(s):

Jinsheng Dong ◽

Alan G Hinnebusch

Keyword(s):

Translation Initiation ◽

Ternary Complex ◽

Start Codon ◽

Yeast Mutant ◽

Initiation Complex ◽

Initiation Factors ◽

Codon Recognition ◽

Open Conformation ◽

Kozak Context

Abstract The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that non-lethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.

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Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

10.1101/098699 ◽

2017 ◽

Author(s):

Jinsheng Dong ◽

Colin Echeverría Aitken ◽

Anil Thakur ◽

Byung-Sik Shin ◽

Jon R. Lorsch ◽

...

Keyword(s):

Translation Initiation ◽

Start Codon ◽

Entry And Exit ◽

Functional Importance ◽

Conserved Residues ◽

Kozak Context ◽

Domains Of Life ◽

Mrna Binding ◽

Yeast Mutants

ABSTRACTThe eukaryotic 43S pre-initiation complex (PIC) bearing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site (“POUT”) to a closed, arrested conformation with TC tightly bound in the “PIN” state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high (Sui-), conferring the Ssu- phenotype. Two such Ssu- substitutions—R116D and R117D—also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PIN state with a UUG:anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC:mRNA contacts at the entry channel, complementing the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.

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