scholarly journals Stable m7G Cap-Distal 5'UTR Hairpin Structure Mediates Distinct 40S and 60S Binding Dynamics

2021 ◽  
Author(s):  
Hongyun Wang ◽  
Anthony Gaba ◽  
Xiaohui Qu
Keyword(s):  
Single Molecule ◽  
Ribosomal Subunit ◽  
Translation System ◽  
Translation Efficiency ◽  
Subunit Interactions ◽  
Ribosomal Subunits ◽  
Initiation Step ◽  
Free Translation ◽  
Hairpin Structures

The 5' untranslated region (UTR) of diverse mRNAs contains secondary structures that can influence protein synthesis by modulating the initiation step of translation. Studies support the ability of these structures to inhibit 40S subunit recruitment and scanning, but the dynamics of ribosomal subunit interactions with mRNA remain poorly understood. Here, we developed a reconstituted Saccharomyces cerevisiae cell-free translation system with fluorescently labeled ribosomal subunits. We applied this extract and single-molecule fluorescence microscopy to monitor, in real time, individual 40S and 60S interactions with mRNAs containing 5' UTR hairpin structures with varying thermostability. In comparison to mRNAs containing no or weak 5' UTR hairpins (ΔG >= -5.4 kcal/mol), mRNAs with stable hairpins (ΔG <= -16.5 kcal/mol) showed reduced numbers of 60S recruitment to mRNA, consistent with the expectation of reduced translation efficiency for such mRNAs. Interestingly, such mRNAs showed increased numbers of 40S recruitment events to individual mRNAs but with shortened duration on mRNA. Correlation analysis showed that these unstable 40S binding events were nonproductive for 60S recruitment. Furthermore, although the mRNA sequence is long enough to accommodate multiple 40S, individual mRNAs are predominantly observed to engage with a single 40S at a time, indicating the sequestering of mRNA 5' end by initiating 40S. Altogether, these observations suggest that stable cap-distal hairpins in 5' UTR reduce initiation and translation efficiency by destabilizing 40S-mRNA interactions and promoting 40S dissociation from mRNA. The premature 40S dissociation frees mRNA 5'-end accessibility for new initiation events, but the increased rate of 40S recruitment is insufficient to compensate for the reduction of initiation efficiency due to premature 40S dissociation. This study provides the first single-molecule kinetic characterization of 40S/60S interactions with mRNA during cap-dependent initiation and the modulation of such interactions by cap-distal 5' UTR hairpin structures.

Characterization of a cell-free translation system from Agrobacterium tumefaciens

1992 ◽  
Vol 20 (4) ◽  
pp. 314S-314S
Author(s):  
J. MIGUEL FERRERAS ◽  
CARLOS ALEGRE ◽  
ROSARIO IGLESIAS ◽  
TOMAS GIRBES
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Translation System ◽  
Free Translation ◽  
A Cell

scholarly journals Precise tuning of bacterial translation initiation by non-equilibrium 5’-UTR unfolding observed in single mRNAs

2021 ◽  
Author(s):  
Sujay Ray ◽  
Shiba Dandpat ◽  
Surajit Chatterjee ◽  
Nils Walter
Keyword(s):  
Single Molecule ◽  
Ribosomal Subunit ◽  
Translation Efficiency ◽  
Messenger Rnas ◽  
Ribosomal Subunit Protein ◽  
Control Translation ◽  
Binding Cleft ◽  
The Stability ◽  
Bacterial Translation Initiation ◽  
Non Equilibrium

Abstract Noncoding, structured 5’- untranslated regions (5’-UTRs) of messenger RNAs (mRNAs) control translation efficiency by forming structures that can either recruit or repel the ribosome. Here we exploit a bacterial, preQ1-sensing translational riboswitch to probe how binding of a small ligand controls binding of the bacterial ribosome to the Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5’-UTR during mRNA accommodation from the standby site to the binding cleft. Ligand binding stabilizes 5’-UTR structure to both antagonize 30S recruitment and accelerate 30S dissociation. Depletion of small ribosomal subunit protein S1, known to resolve structured 5’-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5’-UTR during accommodation provides a mechanistic understanding of translation efficiency.

scholarly journals Autoregulation of Ribosome Biosynthesis by a Translational Response in Fission Yeast

2006 ◽  
Vol 26 (5) ◽  
pp. 1731-1742 ◽  
Author(s):  
François Bachand ◽  
Daniel H. Lackner ◽  
Jürg Bähler ◽  
Pamela A. Silver
Keyword(s):  
Ribosomal Protein ◽  
Fission Yeast ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Transcriptional Level ◽  
Translation Efficiency ◽  
Ribosomal Subunits ◽  
Ribosome Density ◽  
Protein Encoding ◽  
Ribosome Biosynthesis

ABSTRACT Maintaining the appropriate balance between the small and large ribosomal subunits is critical for translation and cell growth. We previously identified the 40S ribosomal protein S2 (rpS2) as a substrate of the protein arginine methyltransferase 3 (RMT3) and reported a misregulation of the 40S/60S ratio in rmt3 deletion mutants of Schizosaccharomyces pombe. For this study, using DNA microarrays, we have investigated the genome-wide biological response of rmt3-null cells to this ribosomal subunit imbalance. Whereas little change was observed at the transcriptional level, a number of genes showed significant alterations in their polysomal-to-monosomal ratios in rmt3Δ mutants. Importantly, nearly all of the 40S ribosomal protein-encoding mRNAs showed increased ribosome density in rmt3 disruptants. Sucrose gradient analysis also revealed that the ribosomal subunit imbalance detected in rmt3-null cells is due to a deficit in small-subunit levels and can be rescued by rpS2 overexpression. Our results indicate that rmt3-null fission yeast compensate for the reduced levels of small ribosomal subunits by increasing the ribosome density, and likely the translation efficiency, of 40S ribosomal protein-encoding mRNAs. Our findings support the existence of autoregulatory mechanisms that control ribosome biosynthesis and translation as an important layer of gene regulation.

scholarly journals Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

2014 ◽  
Vol 112 (2) ◽  
pp. 319-325 ◽  
Author(s):  
Gabriele Fuchs ◽  
Alexey N. Petrov ◽  
Caleb D. Marceau ◽  
Lauren M. Popov ◽  
Jin Chen ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Single Molecule ◽  
Internal Ribosome Entry Site ◽  
Large Scale ◽  
Ribosomal Subunit ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Hcv Ires

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

scholarly journals History of the ribosome and the origin of translation

2015 ◽  
Vol 112 (50) ◽  
pp. 15396-15401 ◽  
Author(s):  
Anton S. Petrov ◽  
Burak Gulen ◽  
Ashlyn M. Norris ◽  
Nicholas A. Kovacs ◽  
Chad R. Bernier ◽  
...  
Keyword(s):  
Comparative Method ◽  
Ribosomal Subunit ◽  
Translation System ◽  
Small Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Origin And Evolution ◽  
Origin Of Translation ◽  
History Of ◽  
Level Model ◽  
Exit Tunnel

We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.

Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide

1981 ◽  
Vol 1 (1) ◽  
pp. 51-57
Author(s):  
T L Helser ◽  
R A Baan ◽  
A E Dahlberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Subunit ◽  
Electrophoretic Analysis ◽  
Initiation Factor ◽  
Ribosomal Subunits ◽  
Messenger Ribonucleic Acid ◽  
Protein Gel ◽  
40S Ribosomal Subunit ◽  
Acid Protein

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.

scholarly journals Functional characterization of ribosomal P1/P2 proteins in human cells

2008 ◽  
Vol 413 (3) ◽  
pp. 527-534 ◽  
Author(s):  
Francisco Martinez-Azorin ◽  
Miguel Remacha ◽  
Juan P. G. Ballesta
Keyword(s):  
Functional Characterization ◽  
Ribosomal Subunit ◽  
Human Cell Line ◽  
Human Cells ◽  
Translation Efficiency ◽  
Ribosomal Stalk ◽  
P Proteins ◽  
Subunit Joining

The ‘stalk’ is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.

scholarly journals Synthesis and Characterization of 18F-Interleukin-8 Using a Cell-Free Translation System and 4-18F-Fluoro-L-Proline

2016 ◽  
Vol 57 (4) ◽  
pp. 634-639 ◽  
Author(s):  
R. Harada ◽  
S. Furumoto ◽  
T. Yoshikawa ◽  
Y. Ishikawa ◽  
K. Shibuya ◽  
...  
Keyword(s):  
Interleukin 8 ◽  
Synthesis And Characterization ◽  
Translation System ◽  
Free Translation ◽  
A Cell

scholarly journals Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide.

1981 ◽  
Vol 1 (1) ◽  
pp. 51-57 ◽  
Author(s):  
T L Helser ◽  
R A Baan ◽  
A E Dahlberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Subunit ◽  
Electrophoretic Analysis ◽  
Initiation Factor ◽  
Ribosomal Subunits ◽  
Messenger Ribonucleic Acid ◽  
Protein Gel ◽  
40S Ribosomal Subunit ◽  
Acid Protein

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.

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