Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

ATP-binding cassette 50 (ABC50; also known as ABCF1) binds to eukaryotic initiation factor 2 (eIF2) and is required for efficient translation initiation. An essential step of this process is accurate recognition and selection of the initiation codon. It is widely accepted that the presence and movement of eIF1, eIF1A and eIF5 are key factors in modulating the stringency of start-site selection, which normally requires an AUG codon in an appropriate sequence context. In the present study, we show that expression of ABC50 mutants, which cannot hydrolyse ATP, decreases general translation and relaxes the discrimination against the use of non-AUG codons at translation start sites. These mutants do not appear to alter the association of key initiation factors to 40S subunits. The stringency of start-site selection can be restored through overexpression of eIF1, consistent with the role of that factor in enhancing stringency. The present study indicates that interfering with the function of ABC50 influences the accuracy of initiation codon selection.

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The Role of eIF1 in Translation Initiation Codon Selection in Caenorhabditiselegans

Genetics ◽

10.1534/genetics.110.121541 ◽

2010 ◽

Vol 186 (4) ◽

pp. 1187-1196 ◽

Cited By ~ 4

Author(s):

Lisa L. Maduzia ◽

Anais Moreau ◽

Nausicaa Poullet ◽

Sebastien Chaffre ◽

Yinhua Zhang

Keyword(s):

Translation Initiation ◽

Initiation Codon ◽

Translation Initiation Codon ◽

Codon Selection

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Alternative mechanisms of initiating translation of mammalian mRNAs

Biochemical Society Transactions ◽

10.1042/bst0331231 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1231-1241 ◽

Cited By ~ 77

Author(s):

R.J. Jackson

Keyword(s):

Site Selection ◽

Mammalian Cells ◽

Ribosomal Subunit ◽

Aggregate Size ◽

Mrna Translation ◽

Initiation Site ◽

Initiation Factor ◽

Viral Mrnas ◽

Initiation Factors ◽

Scanning Mechanism

Of all the steps in mRNA translation, initiation is the one that differs most radically between prokaryotes and eukaryotes. Not only is there no equivalent of the prokaryotic Shine–Dalgarno rRNA–mRNA interaction, but also what requires only three initiation factor proteins (aggregate size ∼125 kDa) in eubacteria needs at least 28 different polypeptides (aggregate >1600 kDa) in mammalian cells, which is actually larger than the size of the 40 S ribosomal subunit. Translation of the overwhelming majority of mammalian mRNAs occurs by a scanning mechanism, in which the 40 S ribosomal subunit, primed for initiation by the binding of several initiation factors including the eIF2 (eukaryotic initiation factor 2)–GTP–MettRNAi complex, is loaded on the mRNA immediately downstream of the 5′-cap, and then scans the RNA in the 5′→3′ direction. On recognition of (usually) the first AUG triplet via base-pairing with the Met-tRNAi anticodon, scanning ceases, triggering GTP hydrolysis and release of eIF2–GDP. Finally, ribosomal subunit joining and the release of the other initiation factors completes the initiation process. This sketchy outline conceals the fact that the exact mechanism of scanning and the precise roles of the initiation factors remain enigmatic. However, the factor requirements for initiation site selection on some viral IRESs (internal ribosome entry sites/segments) are simpler, and investigations into these IRES-dependent mechanisms (particularly picornavirus, hepatitis C virus and insect dicistrovirus IRESs) have significantly enhanced our understanding of the standard scanning mechanism. This article surveys the various alternative mechanisms of initiation site selection on mammalian (and other eukaryotic) cellular and viral mRNAs, starting from the simplest (in terms of initiation factor requirements) and working towards the most complex, which paradoxically happens to be the reverse order of their discovery.

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Translation initiation mediated by RNA looping

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1416883112 ◽

2015 ◽

Vol 112 (4) ◽

pp. 1041-1046 ◽

Cited By ~ 34

Author(s):

Ki Young Paek ◽

Ka Young Hong ◽

Incheol Ryu ◽

Sung Mi Park ◽

Sun Ju Keum ◽

...

Keyword(s):

Reporter Gene ◽

Translation Initiation ◽

Mrna Translation ◽

Encephalomyocarditis Virus ◽

Initiation Codon ◽

Translation Efficiency ◽

Upstream Gene ◽

Translational Initiation ◽

Initiation Factors ◽

Translation Initiation Factors

Eukaryotic translation initiation commences at the initiation codon near the 5′ end of mRNA by a 40S ribosomal subunit, and the recruitment of a 40S ribosome to an mRNA is facilitated by translation initiation factors interacting with the m7G cap and/or poly(A) tail. The 40S ribosome recruited to an mRNA is then transferred to the AUG initiation codon with the help of translation initiation factors. To understand the mechanism by which the ribosome finds an initiation codon, we investigated the role of eIF4G in finding the translational initiation codon. An artificial polypeptide eIF4G fused with MS2 was localized downstream of the reporter gene through MS2-binding sites inserted in the 3′ UTR of the mRNA. Translation of the reporter was greatly enhanced by the eIF4G-MS2 fusion protein regardless of the presence of a cap structure. Moreover, eIF4G-MS2 tethered at the 3′ UTR enhanced translation of the second cistron of a dicistronic mRNA. The encephalomyocarditis virus internal ribosome entry site, a natural translational-enhancing element facilitating translation through an interaction with eIF4G, positioned downstream of a reporter gene, also enhanced translation of the upstream gene in a cap-independent manner. Finally, we mathematically modeled the effect of distance between the cap structure and initiation codon on the translation efficiency of mRNAs. The most plausible explanation for translational enhancement by the translational-enhancing sites is recognition of the initiation codon by the ribosome bound to the ribosome-recruiting sites through “RNA looping.” The RNA looping hypothesis provides a logical explanation for augmentation of translation by enhancing elements located upstream and/or downstream of a protein-coding region.

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The Organizing Principles of Eukaryotic Ribosome Recruitment

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-111042 ◽

2019 ◽

Vol 88 (1) ◽

pp. 307-335 ◽

Cited By ~ 45

Author(s):

Jerry Pelletier ◽

Nahum Sonenberg

Keyword(s):

Protein Synthesis ◽

Initiation Codon ◽

Messenger Rnas ◽

Eukaryotic Ribosome ◽

Initiation Factors ◽

Organizing Principles

The stage at which ribosomes are recruited to messenger RNAs (mRNAs) is an elaborate and highly regulated phase of protein synthesis. Upon completion of this step, a ribosome is positioned at an appropriate initiation codon and primed to synthesize the encoded polypeptide product. In most circumstances, this step commits the ribosome to translate the mRNA. We summarize the knowledge regarding the initiation factors implicated in this activity as well as review different mechanisms by which this process is conducted.

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Selection of start codon during mRNA scanning in eukaryotic translation initiation

10.1101/2020.11.06.371484 ◽

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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Determinants of Initiation Codon Selection during Translation in Mammalian Cells

PLoS ONE ◽

10.1371/journal.pone.0015057 ◽

2010 ◽

Vol 5 (11) ◽

pp. e15057 ◽

Cited By ~ 10

Author(s):

Daiki Matsuda ◽

Vincent P. Mauro

Keyword(s):

Mammalian Cells ◽

Initiation Codon ◽

Codon Selection

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DHX29 reduces leaky scanning through an upstream AUG codon regardless of its nucleotide context

Nucleic Acids Research ◽

10.1093/nar/gkw240 ◽

2016 ◽

Vol 44 (9) ◽

pp. 4252-4265 ◽

Cited By ~ 12

Author(s):

Vera P. Pisareva ◽

Andrey V. Pisarev

Keyword(s):

Mutational Analysis ◽

Ribosomal Subunit ◽

Initiation Factor ◽

Start Codon ◽

Eukaryotic Translation ◽

Initiation Factors ◽

40S Ribosomal Subunit ◽

Nucleotide Context ◽

Eukaryotic Translation Initiation ◽

Codon Selection

Abstract During eukaryotic translation initiation, the 43S preinitiation complex (43S PIC), consisting of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA scans mRNA to find an appropriate start codon. Key roles in the accuracy of initiation codon selection belong to eIF1 and eIF1A, whereas the mammalian-specific DHX29 helicase substantially contributes to ribosomal scanning of structured mRNAs. Here, we show that DHX29 stimulates the recognition of the AUG codon but not the near-cognate CUG codon regardless of its nucleotide context during ribosomal scanning. The stimulatory effect depends on the contact between DHX29 and eIF1A. The unique DHX29 N-terminal domain binds to the ribosomal site near the mRNA entrance, where it contacts the eIF1A OB domain. UV crosslinking assays revealed that DHX29 may rearrange eIF1A and eIF2α in key nucleotide context positions of ribosomal complexes. Interestingly, DHX29 impedes the 48S initiation complex formation in the absence of eIF1A perhaps due to forming a physical barrier that prevents the 43S PIC from loading onto mRNA. Mutational analysis allowed us to split the mRNA unwinding and codon selection activities of DHX29. Thus, DHX29 is another example of an initiation factor contributing to start codon selection.

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Roles of helix H69 of 23S rRNA in translation initiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507703112 ◽

2015 ◽

Vol 112 (37) ◽

pp. 11559-11564 ◽

Cited By ~ 9

Author(s):

Qi Liu ◽

Kurt Fredrick

Keyword(s):

Translation Initiation ◽

Start Codon ◽

23S Rrna ◽

Initiator Trna ◽

Lethal Phenotype ◽

Initiation Factors ◽

Dominant Lethal ◽

Initiation Of Translation ◽

Codon Selection ◽

Subunit Joining

Initiation of translation involves the assembly of a ribosome complex with initiator tRNA bound to the peptidyl site and paired to the start codon of the mRNA. In bacteria, this process is kinetically controlled by three initiation factors—IF1, IF2, and IF3. Here, we show that deletion of helix H69 (∆H69) of 23S rRNA allows rapid 50S docking without concomitant IF3 release and virtually eliminates the dependence of subunit joining on start codon identity. Despite this, overall accuracy of start codon selection, based on rates of formation of elongation-competent 70S ribosomes, is largely uncompromised in the absence of H69. Thus, the fidelity function of IF3 stems primarily from its interplay with initiator tRNA rather than its anti-subunit association activity. While retaining fidelity, ∆H69 ribosomes exhibit much slower rates of overall initiation, due to the delay in IF3 release and impedance of an IF3-independent step, presumably initiator tRNA positioning. These findings clarify the roles of H69 and IF3 in the mechanism of translation initiation and explain the dominant lethal phenotype of the ∆H69 mutation.

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