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

Genetics ◽  
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.

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 The β-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jyothsna Visweswaraiah ◽  
Yvette Pittman ◽  
Thomas E Dever ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Exit Channel ◽  
Hairpin Loop ◽  
Leaky Scanning ◽  
Initiation Factors ◽  
Codon Recognition

The eukaryotic 43S pre-initiation complex bearing tRNAiMet scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the β-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2∙GTP∙Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in β-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred ‘leaky-scanning’ of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the β-strand-1 substitution greatly destabilized the ‘PIN’ conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in β-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 β-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition.

scholarly journals Interface between 40S exit channel protein uS7/Rps5 and eIF2α modulates start codon recognition in vivo

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jyothsna Visweswaraiah ◽  
Alan G Hinnebusch
Keyword(s):  
Ternary Complex ◽  
Start Codon ◽  
Exit Channel ◽  
Initiation Complex ◽  
Channel Protein ◽  
Codon Recognition ◽  
Closed State ◽  
Complex Increase

The eukaryotic pre-initiation complex (PIC) bearing the eIF2·GTP·Met-tRNAiMet ternary complex (TC) scans the mRNA for an AUG codon in favorable context. AUG recognition evokes rearrangement of the PIC from an open, scanning to a closed, arrested conformation. Cryo-EM reconstructions of yeast PICs suggest remodeling of the interface between 40S protein Rps5/uS7 and eIF2α between open and closed states; however, its importance was unknown. uS7 substitutions disrupting eIF2α contacts favored in the open complex increase initiation at suboptimal sites, and uS7-S223D stabilizes TC binding to PICs reconstituted with a UUG start codon, indicating inappropriate rearrangement to the closed state. Conversely, uS7-D215 substitutions, perturbing uS7-eIF2α interaction in the closed state, confer the opposite phenotypes of hyperaccuracy and (for D215L) accelerated TC dissociation from reconstituted PICs. Thus, remodeling of the uS7/eIF2α interface appears to stabilize first the open, and then the closed state of the PIC to promote accurate AUG selection in vivo.

scholarly journals Eukaryotic aspects of translation initiation brought into focus

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160186 ◽  
Author(s):  
Christopher H. S. Aylett ◽  
Nenad Ban
Keyword(s):  
Translation Initiation ◽  
Structural Information ◽  
Start Codon ◽  
Translation Regulation ◽  
Initiation Factors ◽  
Codon Recognition ◽  
Initiation Of Translation ◽  
Control Translation ◽  
Additional Level ◽  
Subunit Joining

In all organisms, mRNA-directed protein synthesis is catalysed by ribosomes. Although the basic aspects of translation are preserved in all kingdoms of life, important differences are found in the process of translation initiation, which is rate-limiting and the most important step for translation regulation. While great strides had been taken towards a complete structural understanding of the initiation of translation in eubacteria, our understanding of the eukaryotic process, which includes numerous eukaryotic-specific initiation factors, was until recently limited owing to a lack of structural information. In this review, we discuss recent results in the field that provide an increasingly complete molecular description of the eukaryotic initiation process. The structural snapshots obtained using a range of methods now provide insights into the architecture of the initiation complex, start-codon recognition by the initiator tRNA and the process of subunit joining. Future advances will require both higher-resolution insights into previously characterized complexes and mapping of initiation factors that control translation on an additional level by interacting only peripherally or transiently with ribosomal subunits. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

2000 ◽  
Vol 20 (19) ◽  
pp. 7183-7191 ◽  
Author(s):  
Sang Ki Choi ◽  
DeAnne S. Olsen ◽  
Antonina Roll-Mecak ◽  
Agnes Martung ◽  
Keith L. Remo ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Sequence Similarity ◽  
Start Codon ◽  
Amino Acid Sequence Similarity ◽  
Physical Interaction ◽  
Binding Assays ◽  
Initiation Factors ◽  
Translation Initiation Factors

ABSTRACT To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

scholarly journals Structural Changes Enable Start Codon Recognition by the Eukaryotic Translation Initiation Complex

Cell ◽  
2014 ◽  
Vol 159 (3) ◽  
pp. 597-607 ◽  
Author(s):  
Tanweer Hussain ◽  
Jose L. Llácer ◽  
Israel S. Fernández ◽  
Antonio Munoz ◽  
Pilar Martin-Marcos ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Structural Changes ◽  
Start Codon ◽  
Initiation Complex ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
Eukaryotic Translation Initiation

scholarly journals eIF3 and Its mRNA-Entry-Channel Arm Contribute to the Recruitment of mRNAs With Long 5′-Untranslated Regions

2022 ◽  
Vol 8 ◽  
Author(s):  
Andrei Stanciu ◽  
Juncheng Luo ◽  
Lucy Funes ◽  
Shanya Galbokke Hewage ◽  
Colin Echeverría Aitken
Keyword(s):  
Translation Initiation ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Start Codon ◽  
Untranslated Regions ◽  
Initiation Factors ◽  
Codon Recognition ◽  
Entire Complex ◽  
The Individual

Translation initiation in eukaryotes is a multi-step pathway and the most regulated phase of translation. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex of the translation initiation factors, and it contributes to events throughout the initiation pathway. In particular, eIF3 appears to play critical roles in mRNA recruitment. More recently, eIF3 has been implicated in driving the selective translation of specific classes of mRNAs. However, unraveling the mechanism of these diverse contributions—and disentangling the roles of the individual subunits of the eIF3 complex—remains challenging. We employed ribosome profiling of budding yeast cells expressing two distinct mutations targeting the eIF3 complex. These mutations either disrupt the entire complex or subunits positioned near the mRNA-entry channel of the ribosome and which appear to relocate during or in response to mRNA binding and start-codon recognition. Disruption of either the entire eIF3 complex or specific targeting of these subunits affects mRNAs with long 5′-untranslated regions and whose translation is more dependent on eIF4A, eIF4B, and Ded1 but less dependent on eIF4G, eIF4E, and PABP. Disruption of the entire eIF3 complex further affects mRNAs involved in mitochondrial processes and with structured 5′-untranslated regions. Comparison of the suite of mRNAs most sensitive to both mutations with those uniquely sensitive to disruption of the entire complex sheds new light on the specific roles of individual subunits of the eIF3 complex.

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