scholarly journals A short translational ramp determines the efficiency of protein synthesis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Manasvi Verma ◽  
Junhong Choi ◽  
Kyle A. Cottrell ◽  
Zeno Lavagnino ◽  
Erica N. Thomas ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Translation Initiation ◽  
Translational Control ◽  
Critical Role ◽  
Sequence Motifs ◽  
Control Of Gene Expression ◽  
Codon Positions

AbstractTranslation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.

scholarly journals Short translational ramp determines efficiency of protein synthesis

2019 ◽  
Author(s):  
Manasvi Verma ◽  
Junhong Choi ◽  
Kyle A. Cottrell ◽  
Zeno Lavagnino ◽  
Erica N. Thomas ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Single Molecule ◽  
Translation Initiation ◽  
Protein Translation ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Sequence Motifs ◽  
Peptide Bond Formation

AbstractIt is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence1–4. These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5’ translational ramp, likely contributes to the efficiency of protein synthesis 5–9. Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias6,9, the rate of translation initiation10–15, mRNA and ribosome structure 11,12,14,16–18, or retention of initiation factors during early elongation events 19. Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4th or 5th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

scholarly journals Folding and Persistence Time of Intramolecular G-Quadruplexes Transiently Embedded in a DNA duplex

2021 ◽  
Author(s):  
Phong Lan Thao Tran ◽  
Martin Rieu ◽  
Samar Hodeib ◽  
Alexandra Joubert ◽  
Jimmy Ouellet ◽  
...  
Keyword(s):  
Single Molecule ◽  
Critical Role ◽  
Genetic Regulation ◽  
Regulatory Elements ◽  
Promoter Regions ◽  
Folding Rate ◽  
Persistence Time ◽  
G4 Dna

ABSTRACTG-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. While many in-vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in-vivo in double-stranded DNA regions, where their formation is challenged by pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4 in vivo. To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than in telomeric regions. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence increased. Our assay opens new perspectives for the measurement of G4 dynamics, which is critical to understand their role in genetic regulation.

β-Oxidation of free fatty acids is required to maintain translational control of protein synthesis in heart

2002 ◽  
Vol 283 (6) ◽  
pp. E1144-E1150 ◽  
Author(s):  
Stephen J. Crozier ◽  
Douglas R. Bolster ◽  
Ali K. Reiter ◽  
Scot R. Kimball ◽  
Leonard S. Jefferson
Keyword(s):  
Fatty Acids ◽  
Protein Synthesis ◽  
Free Fatty Acids ◽  
Translation Initiation ◽  
Translational Control ◽  
Mrna Translation ◽  
Nucleotide Exchange ◽  
Energy Status ◽  
Α Subunit

The study described herein investigated the role of free fatty acids (FFAs) in the maintenance of protein synthesis in vivo in rat cardiac and skeletal muscle. Suppression of FFA β-oxidation by methyl palmoxirate caused a marked reduction in protein synthesis in the heart. The effect on protein synthesis was mediated in part by changes in the function of eukaryotic initiation factors (eIFs) involved in the initiation of mRNA translation. The guanine nucleotide exchange activity of eIF2B was repressed, phosphorylation of the α-subunit of eIF2 was enhanced, and phosphorylation of eIF4E-binding protein-1 and ribosomal protein S6 kinase was reduced. Similar changes in protein synthesis and translation initiation were not observed in the gastrocnemius following treatment with methyl palmoxirate. In heart, repressed β-oxidation of FFA correlated, as demarcated by changes in the ATP/AMP ratio and phosphorylation of AMP-activated kinase, with alterations in the energy status of the tissue. Therefore, the activation state of signal transduction pathways that are responsive to cellular energy stress represents one mechanism whereby translation initiation may be regulated in cardiac muscle.

scholarly journals (p)ppGpp directly regulates translation initiation during entry into quiescence

2019 ◽  
Author(s):  
Simon Diez ◽  
Jaewook Ryu ◽  
Kelvin Caban ◽  
Ruben L. Gonzalez ◽  
Jonathan Dworkin
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Extended Period ◽  
Initiation Factor ◽  
Allosteric Activation ◽  
Initiation Factor 2 ◽  
Low Levels ◽  
Mechanistic Basis

SummaryMany bacteria exist in a state of metabolic quiescence where they must minimize energy consumption so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for downregulation during the transition from growth to quiescence. We find that whenBacillus subtilisexits growth, a subpopulation of cells emerges with very low levels of protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp inhibits protein synthesisin vivoandin vitroby preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is an authenticin vivotarget of (p)ppGpp during the entry into quiescence, thus providing a mechanistic basis for the observed attenuation of protein synthesis.

scholarly journals The Ubiquitin-Dependent Targeting Pathway in Saccharomyces cerevisiae Plays a Critical Role in Multiple Chromatin Assembly Regulatory Steps

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 615-632 ◽  
Author(s):  
Troy A A Harkness ◽  
Gerald F Davies ◽  
Vijay Ramaswamy ◽  
Terra G Arnason
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translational Control ◽  
Regulatory Networks ◽  
Critical Role ◽  
Chromatin Assembly ◽  
Temperature Sensitive ◽  
Anaphase Promoting Complex ◽  
Free Media

Abstract In a screen designed to isolate Saccharomyces cerevisiae strains defective for in vitro chromatin assembly, two temperature-sensitive (ts) mutants were obtained: rmc1 and rmc3 (remodeling of chromatin). Cloning of RMC1 and RMC3 revealed a broad role for the ubiquitin-dependent targeting cascade as the ubiquitin-protein ligases (E3s), the anaphase promoting complex (APC; RMC1 encodes APC5) and Rsp5p, respectively, were identified. Genetic studies linked the rmc1/apc5 chromatin assembly defect to APC function: rmc1/apc5 genetically interacted with apc9Δ, apc10Δ, and cdc26Δ mutants. Furthermore, phenotypes associated with the rmc1/apc5 allele were consistent with defects in chromatin metabolism and in APC function: (i) UV sensitivity, (ii) plasmid loss, (iii) accumulation of G2/M cells, and (iv) suppression of the ts defect by growth on glucose-free media and by expression of ubiquitin. On the other hand, the multifunctional E3, Rsp5p, was shown to be required for both in vitro and in vivo chromatin assembly, as well as for the proper transcriptional and translational control of at least histone H3. The finding that the distinctly different E3 enzymes, APC and Rsp5p, both play roles in regulating chromatin assembly highlight the depth of the regulatory networks at play. The significance of these findings will be discussed.

scholarly journals An in vitro single-molecule assay for eukaryotic cap-dependent translation initiation kinetics

2019 ◽  
Author(s):  
Hongyun Wang ◽  
Lexi Sun ◽  
Anthony Gaba ◽  
Xiaohui Qu
Keyword(s):  
Single Molecule ◽  
Translation Initiation ◽  
Translational Control ◽  
Budding Yeast ◽  
Chain Elongation ◽  
Time Range ◽  
Control Mechanisms ◽  
General Applicability ◽  
Synthesis Time

Abstract Eukaryotic mRNAs are predominantly translated via the cap-dependent pathway. Initiation is a rate-limiting step in cap-dependent translation and is the main target of translational control mechanisms. There is a lack of high-resolution techniques for characterizing the cap-dependent initiation kinetics. Here, we report an in vitro single-molecule assay that allows characterization of both initiation and peptide chain elongation kinetics for cap-dependent translation. Surprisingly, the histogram of the first-round initiation time is highly asymmetrical and spans a large time range that is several-fold greater than the average peptide synthesis time in translation reactions with a firefly luciferase-encoding mRNA. Both the histogram and single-molecule trajectories reveal an unexpected high-degree of asynchrony in translation activity between mRNA molecules. Furthermore, by inserting a small stem-loop (ΔG = −4.8 kcal/mol) in the middle of the mRNA 5′ untranslated region (UTR), our assay robustly detects small changes in budding yeast initiation kinetics, which could not be resolved by bulk luminescence kinetics. Lastly, we demonstrate the general applicability of this assay to distinct cell-free translation systems by using extracts prepared from budding yeast, wheat germ, and rabbit reticulocyte lysates. This assay should facilitate mechanistic studies of eukaryotic cap-dependent translation initiation and translational control.

scholarly journals Cleavage of Poly(A)-Binding Protein by Coxsackievirus 2A Protease In Vitro and In Vivo: Another Mechanism for Host Protein Synthesis Shutoff?

1999 ◽  
Vol 73 (1) ◽  
pp. 709-717 ◽  
Author(s):  
Vaishali Kerekatte ◽  
Brett D. Keiper ◽  
Cornel Badorff ◽  
Aili Cai ◽  
Kirk U. Knowlton ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Viral Protein ◽  
Binding Protein ◽  
Host Protein ◽  
Viral Protein Synthesis ◽  
2A Protease ◽  
Host Protein Synthesis

ABSTRACT Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments withXenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at482VANTSTQTM↓GPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.

Inducible Silencing Of eIF4E Using a Tet-On System Results In Myeloma Growth In Vivo That Correlates With eIF4E Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3164-3164
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Jordan M. Schecter ◽  
Caisheng Lu ◽  
Markus Y. Mapara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Tumor Growth ◽  
Critical Role ◽  
Myeloma Cell ◽  
Control Shrna

Abstract Introduction Overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro. Methods and Results We stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P<0.01) cell growth accompanied by a decrease of c-myc, cyclin D1, C/EBP beta and IRF4 all critical for myeloma cell growth. Cell cycle analysis revealed increased cells population in G0/G1 phase (62% vs 80%) in doxycycline-induced eIF4E shRNA cells with a significant reduction (P<0.001) of clonogenic tumor growth reflected by a decrease in colony numbers (27.6 ± 4.2 vs 5.3 ± 3.4) and size. To determine the role of high expression of eIF4E in MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the inducible U266-Tet-CT-shRNA and U266-Tet-eIF4E-shRNA cells. In contrast to vehicle or doxycycline-treated control shRNA tumors, doxycycline treated animals bearing U266-Tet-eIF4E-shRNA xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 21 days. The transient inhibition of tumor growth correlated with the transient doxycycline-induced eIF4E knockdown further confirming the critical role of eIF4E. Immunohistochemical staining of tumors confirmed the decreased of eIF4E expression in doxycycline-treated mice bearing U266-Tet-eIF4E-shRNA tumors compared with tumors of vehicle-treated or non-doxycyclin treated mice. Conclusion Here we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.

scholarly journals Dendritic BC1 RNA in translational control mechanisms

2005 ◽  
Vol 171 (5) ◽  
pp. 811-821 ◽  
Author(s):  
Huidong Wang ◽  
Anna Iacoangeli ◽  
Daisy Lin ◽  
Keith Williams ◽  
Robert B. Denman ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translational Control ◽  
Fragile X ◽  
Initiation Factor ◽  
Control Mechanisms ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Specific Effector

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3′ domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3′ BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors.

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