scholarly journals The C-Terminal Domain of Eukaryotic Initiation Factor 5 Promotes Start Codon Recognition by Its Dynamic Interplay with eIF1 and eIF2β

Cell Reports ◽  
2012 ◽  
Vol 1 (6) ◽  
pp. 689-702 ◽  
Author(s):  
Rafael E. Luna ◽  
Haribabu Arthanari ◽  
Hiroyuki Hiraishi ◽  
Jagpreet Nanda ◽  
Pilar Martin-Marcos ◽  
...  
Keyword(s):  
Initiation Factor ◽  
Start Codon ◽  
Eukaryotic Initiation Factor ◽  
Codon Recognition ◽  
Terminal Domain ◽  
Dynamic Interplay

scholarly journals The complete, functional and dynamic cycle of the bacterial Initiation Factor 3

2019 ◽  
Author(s):  
Jose A. Nakamoto ◽  
Roberto Spurio ◽  
Andrey L. Konevega ◽  
Attilio Fabbretti ◽  
Pohl Milón
Keyword(s):  
Complex Formation ◽  
Translation Initiation ◽  
Binding Sites ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Start Codon ◽  
Initiation Complex ◽  
Terminal Domain ◽  
Dynamic Interplay ◽  
Dynamic Cycle

AbstractInitiation factor 3 (IF3) is an essential protein that enhances the fidelity and speed of bacterial initiation of mRNA translation. The dynamic interplay between the two independent IF3 domains, their alternative binding sites, and the mechanism that ensures translation initiation fidelity remains elusive. Here, we show that the functional positioning of IF3 domains occurs at velocities ranging over two orders of magnitude, driven by each 30S initiation ligand. IF1 and IF2 rapidly promote the accommodation of IF3 on the 30S platform with the C-terminal domain moving towards the P site. Reversion of this movement is triggered by decoding the mRNA start codon and rate limits translation initiation. Binding of the tRNA results in the concomitant accommodation of the N-terminal domain of IF3, largely dependent on the mRNA and initiator tRNA. 70S initiation complex formation promotes the closing and dissociation of IF3, recycling the factor for a new round of translation initiation. Altogether our results unveil the kinetic spectrum of IF3 conformations and highlight fundamental movements of the factor that ensure accurate translation initiation.

scholarly journals Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)

2013 ◽  
Vol 289 (3) ◽  
pp. 1704-1722 ◽  
Author(s):  
Fujun Zhou ◽  
Sarah E. Walker ◽  
Sarah F. Mitchell ◽  
Jon R. Lorsch ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Rna Recognition Motif ◽  
Conserved Motifs ◽  
Terminal Domain ◽  
Sequence Elements

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

scholarly journals Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

2019 ◽  
Author(s):  
Susan Wagner ◽  
Anna Herrmannová ◽  
Vladislava Hronová ◽  
Neelam Sen ◽  
Ross D. Hannan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Translational Control ◽  
Mammalian Cells ◽  
Regulation Of Gene Expression ◽  
Ribosome Profiling ◽  
Initiation Factor ◽  
Start Codon ◽  
Codon Recognition ◽  
Initiation Phase ◽  
Gene Expression Studies

SUMMARYTranslational control targeting mainly the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast Translational Complex Profile sequencing (TCP-seq), related to ribosome profiling, and adopted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ses) in addition to 80S ribosomes (80Ses), revealed that mammalian and yeast 40Ses distribute similarly across 5’UTRs indicating considerable evolutionary conservation. We further developed a variation called Selective TCP-seq (Sel-TCP-seq) enabling selection for 40Ses and 80Ses associated with an immuno-targeted factor in yeast and human. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5’UTRs with scanning 40Ses to successively dissociate upon start codon recognition. Manifesting the Sel-TCP-seq versatility for gene expression studies, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

scholarly journals Functional Characterization of the Role of the N-terminal Domain of the c/Nip1 Subunit of Eukaryotic Initiation Factor 3 (eIF3) in AUG Recognition

2012 ◽  
Vol 287 (34) ◽  
pp. 28420-28434 ◽  
Author(s):  
Martina Karásková ◽  
Stanislava Gunišová ◽  
Anna Herrmannová ◽  
Susan Wagner ◽  
Vanda Munzarová ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Terminal Domain

scholarly journals Structure of a human 48S translational initiation complex

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. 1220-1227 ◽  
Author(s):  
Jailson Brito Querido ◽  
Masaaki Sokabe ◽  
Sebastian Kraatz ◽  
Yuliya Gordiyenko ◽  
J. Mark Skehel ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Messenger Rna ◽  
Initiation Factor ◽  
Start Codon ◽  
Exit Channel ◽  
Eukaryotic Initiation Factor ◽  
Initiation Complex ◽  
Translational Initiation ◽  
Cryo Electron Microscopy ◽  
Cap Binding Complex

A key step in translational initiation is the recruitment of the 43S preinitiation complex by the cap-binding complex [eukaryotic initiation factor 4F (eIF4F)] at the 5′ end of messenger RNA (mRNA) to form the 48S initiation complex (i.e., the 48S). The 48S then scans along the mRNA to locate a start codon. To understand the mechanisms involved, we used cryo–electron microscopy to determine the structure of a reconstituted human 48S. The structure reveals insights into early events of translation initiation complex assembly, as well as how eIF4F interacts with subunits of eIF3 near the mRNA exit channel in the 43S. The location of eIF4F is consistent with a slotting model of mRNA recruitment and suggests that downstream mRNA is unwound at least in part by being “pulled” through the 40S subunit during scanning.

scholarly journals Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex

2014 ◽  
Vol 42 (15) ◽  
pp. 9623-9640 ◽  
Author(s):  
Adesh K Saini ◽  
Jagpreet S Nanda ◽  
Pilar Martin-Marcos ◽  
Jinsheng Dong ◽  
Fan Zhang ◽  
...  
Keyword(s):  
Critical Role ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Preinitiation Complex ◽  
Subsequent Work ◽  
Gtp Hydrolysis ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
Efficient Suppression

Abstract eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNAiMet ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2·GDP·Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui− mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection invivo.

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