scholarly journals Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

2021 ◽  
Author(s):  
Jinfan Wang ◽  
Carlos Alvarado ◽  
Byung-Sik Shin ◽  
Jonathan Bohlen ◽  
Thomas E. Dever ◽  
...  
Keyword(s):  
Direct Observation ◽  
Single Molecule ◽  
Translational Regulation ◽  
Atp Hydrolysis ◽  
Start Codon ◽  
Rna Helicases ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Mrna Binding ◽  
Subunit Joining

AbstractHow the eukaryotic 43S preinitiation complex scans along the 5′ untranslated region (5′UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. Once engaged with the mRNA, 43S scanning occurs at >100 nucleotides per second, independent of multiple cycles of ATP-hydrolysis by RNA helicases. The scanning ribosomes can proceed through RNA secondary structures, but 5′UTR hairpin sequences near start codons drive scanning ribosomes at start codons back in the 5′ direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5′UTR structures and upstream near-cognate start codons.One Sentence SummaryDirect observation of scanning eukaryotic ribosomes establishes a quantitative framework of scanning and its regulation.

scholarly journals Linking the 3′ Poly(A) Tail to the Subunit Joining Step of Translation Initiation: Relations of Pab1p, Eukaryotic Translation Initiation Factor 5B (Fun12p), and Ski2p-Slh1p

2001 ◽  
Vol 21 (15) ◽  
pp. 4900-4908 ◽  
Author(s):  
Anjanette Searfoss ◽  
Thomas E. Dever ◽  
Reed Wickner
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rna Helicases ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Subunit Joining

ABSTRACT The 3′ poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)+ mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)+ mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.

scholarly journals Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

2021 ◽  
Vol 118 (6) ◽  
pp. e2017715118
Author(s):  
Christopher P. Lapointe ◽  
Rosslyn Grosely ◽  
Alex G. Johnson ◽  
Jinfan Wang ◽  
Israel S. Fernández ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Start Codon ◽  
Messenger Rnas ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
40S Ribosomal Subunit ◽  
Eukaryotic Translation Initiation Factors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

scholarly journals Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

2007 ◽  
Vol 27 (6) ◽  
pp. 2384-2397 ◽  
Author(s):  
Jeanne M. Fringer ◽  
Michael G. Acker ◽  
Christie A. Fekete ◽  
Jon R. Lorsch ◽  
Thomas E. Dever
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Cell Extracts ◽  
Eukaryotic Translation Initiation ◽  
Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

scholarly journals eIF1 Controls Multiple Steps in Start Codon Recognition during Eukaryotic Translation Initiation

2009 ◽  
Vol 394 (2) ◽  
pp. 268-285 ◽  
Author(s):  
Jagpreet S. Nanda ◽  
Yuen-Nei Cheung ◽  
Julie E. Takacs ◽  
Pilar Martin-Marcos ◽  
Adesh K. Saini ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Start Codon ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
Eukaryotic Translation Initiation

scholarly journals Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

2021 ◽  
Author(s):  
Silvia Hormeno ◽  
Oliver J Wilkinson ◽  
Clara Aicart-Ramos ◽  
Sahiti Kuppa ◽  
Edwin Antony ◽  
...  
Keyword(s):  
Dna Replication ◽  
Direct Observation ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Motor Protein ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Dna Loops ◽  
Single Stranded Dna ◽  
A Site

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ~20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

Mechanism of ribosomal subunit joining during eukaryotic translation initiation

2008 ◽  
Vol 36 (4) ◽  
pp. 653-657 ◽  
Author(s):  
Michael G. Acker ◽  
Jon R. Lorsch
Keyword(s):  
Translation Initiation ◽  
Control Point ◽  
Ribosomal Subunit ◽  
Eukaryotic Translation ◽  
Translational Machinery ◽  
Biophysical Studies ◽  
Eukaryotic Translation Initiation ◽  
A Cell ◽  
Subunit Joining ◽  
Compare And Contrast

Decades of research have yielded significant insight into the mechanism by which a cell translates an mRNA into the encoded protein. However many of the molecular details of the process remain a mystery. Translation initiation is an important control point in gene expression, and misregulation can lead to diseases such as cancer. A better understanding of the mechanism of translation initiation is imperative for the development of novel therapeutic agents. Recently, a combination of genetic, biochemical and biophysical studies has begun to shed light on how, at a molecular level, the translational machinery initiates protein synthesis. In the present review, we briefly compare and contrast the initiation pathways utilized by bacteria, archaea and eukaryotes, and then focus on translation initiation in eukaryotes and recent advances in our understanding of the subunit joining step of the process.

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