scholarly journals Suppression of Ribosomal Pausing by eIF5A Is Necessary to Maintain the Fidelity of Start Codon Selection

Cell Reports ◽  
2019 ◽  
Vol 29 (10) ◽  
pp. 3134-3146.e6 ◽  
Author(s):  
Hema Manjunath ◽  
He Zhang ◽  
Frederick Rehfeld ◽  
Jaeil Han ◽  
Tsung-Cheng Chang ◽  
...  
Keyword(s):  
Start Codon ◽  
Codon Selection ◽  
Ribosomal Pausing

scholarly journals eIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex

2020 ◽  
Vol 48 (18) ◽  
pp. 10280-10296
Author(s):  
Anil Thakur ◽  
Swati Gaikwad ◽  
Anil K Vijjamarri ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Preinitiation Complex ◽  
Closed Conformation ◽  
Open Conformation ◽  
Kozak Context ◽  
Codon Selection

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.

scholarly journals Cryo-EM study of start codon selection during archaeal translation initiation

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Pierre-Damien Coureux ◽  
Christine Lazennec-Schurdevin ◽  
Auriane Monestier ◽  
Eric Larquet ◽  
Lionel Cladière ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Start Codon ◽  
Codon Selection

scholarly journals eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex

2018 ◽  
Vol 115 (18) ◽  
pp. E4159-E4168 ◽  
Author(s):  
Anil Thakur ◽  
Alan G. Hinnebusch
Keyword(s):  
Initiation Factor ◽  
Start Codon ◽  
Preinitiation Complex ◽  
Initiation Factor 2 ◽  
D Loop ◽  
Double Substitution ◽  
Codon Selection ◽  
Loop 2

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNAi) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a “POUT” state to a closed conformation with TC more tightly bound in a “PIN” state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the PIN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the PIN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi. We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2–D loop interactions specifically impede Met-tRNAi accommodation in the PIN state without influencing the POUT mode of TC binding; and Arg substitutions convert the Loop 2–tRNAi clash to an electrostatic attraction that stabilizes PIN and enhances selection of poor start codons in vivo.

A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

2004 ◽  
Vol 36 (4) ◽  
pp. 259-268 ◽  
Author(s):  
Li Yang ◽  
Jiang Chen ◽  
Catherine C. Y. Chang ◽  
Xin-Ying Yang ◽  
Zhen-Zhen Wang ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Start Codon ◽  
Loop Structure ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Codon Selection ◽  
Mutation Analyses ◽  
Enzymatic Activity Assay ◽  
Selection Of

Abstract Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397–1399 and AUG1415–1417, at 5′-terminus of the open reading frame (ORF, nt 1397–3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397–1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5′-ORF-AUG1397–1399 as a main start codon, in addition to upstream nucleotide A in the –3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.

scholarly journals Why is start codon selection so precise in eukaryotes?

Translation ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. e28387 ◽  
Author(s):  
Katsura Asano
Keyword(s):  
Start Codon ◽  
Codon Selection

scholarly journals Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5

2011 ◽  
Vol 40 (7) ◽  
pp. 2898-2906 ◽  
Author(s):  
Gary Loughran ◽  
Matthew S. Sachs ◽  
John F. Atkins ◽  
Ivaylo P. Ivanov
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Codon Selection

scholarly journals Bi-directional ribosome scanning controls the stringency of start codon selection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yifei Gu ◽  
Yuanhui Mao ◽  
Longfei Jia ◽  
Leiming Dong ◽  
Shu-Bing Qian
Keyword(s):  
Translation Initiation ◽  
Current Knowledge ◽  
Start Codon ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Translation ◽  
Operational Mechanism ◽  
Codon Recognition ◽  
Codon Selection ◽  
Selection Of

AbstractThe fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5ʹ→3ʹ migration of the pre-initiation complex (PIC) along the 5ʹ UTR. In probing translation initiation from ultra-short 5ʹ UTR, we report that an AUG triplet near the 5ʹ end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5ʹ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5ʹ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.

scholarly journals Selection of start codon during mRNA scanning in eukaryotic translation initiation

2020 ◽  
Author(s):  
Ipsita Basu ◽  
Biswajit Gorai ◽  
Thyageshwar Chandran ◽  
Prabal K. Maiti ◽  
Tanweer Hussain
Keyword(s):  
High Speed ◽  
Critical Role ◽  
Free Energy Calculations ◽  
Start Codon ◽  
Translational Initiation ◽  
Initiation Factors ◽  
Closed State ◽  
Open Conformation ◽  
Codon Selection ◽  
Selection Of

AbstractDuring translational initiation in eukaryotes, the small ribosomal subunit forms a 48S preinitiation complex (PIC) with initiation factors. The 48S PIC binds to the 5’ end of mRNA and inspects long untranslated region (UTR) for the presence of the start codon (AUG). Accurate and high speed of scanning 5’ UTR and subsequent selection of the correct start codon are crucial for protein synthesis. However, the conformational state of 48S PIC required for inspecting every codon is not clearly understood. Whether the scanning or open conformation of 48S PIC can accurately select the cognate start codon over near/non-cognate codons, or this discrimination is carried out only in the scanning-arrested or closed conformation of 48S PIC. Here, using atomistic molecular dynamics (MD) simulations and free energy calculations, we show that the scanning conformation of 48S PIC can reject all but 4 of the 63 non-AUG codons. Among nine near-cognate codons with a single mismatch, only codons with a first position mismatch (GUG, CUG and UUG) or a pyrimidine mismatch at the second position (ACG) are not discriminated by scanning state of 48S PIC. In contrast, any mismatch in the third position is rejected. Simulations runs in absence of one or more eukaryotic initiation factors (eIF1, eIF1+eIF1A, eIF2ɑ or eIF2β) from the system show critical role of eIF1 and eIF2ɑ in start codon selection. The structural analysis indicates that tRNAi dynamics at the widened P site of 48S open state drives codon selection. Further, a stable codon: anticodon interaction prepares the PIC to transit to the closed state. Overall, we provide insights into the selection of start codon during scanning and how the open conformation of 48S PIC can scan long 5’ UTRs with accuracy and high speed without the requirement of sampling the closed state for every codon.

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