Short translational ramp determines efficiency of protein synthesis

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.

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A short translational ramp determines the efficiency of protein synthesis

Nature Communications ◽

10.1038/s41467-019-13810-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 17

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.

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Amino acid sequence of the human protein synthesis initiation factor eIF-4 gamma

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)50080-6 ◽

1992 ◽

Vol 267 (32) ◽

pp. 23226-23231

Author(s):

R Yan ◽

W Rychlik ◽

D Etchison ◽

R.E. Rhoads

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Amino Acid Sequence ◽

Human Protein ◽

Initiation Factor ◽

Protein Synthesis Initiation

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Codon usage of highly expressed genes affects proteome-wide translation efficiency

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719375115 ◽

2018 ◽

Vol 115 (21) ◽

pp. E4940-E4949 ◽

Cited By ~ 57

Author(s):

Idan Frumkin ◽

Marc J. Lajoie ◽

Christopher J. Gregg ◽

Gil Hornung ◽

George M. Church ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Codon Usage ◽

Codon Usage Bias ◽

Protein Translation ◽

Translation Efficiency ◽

Synonymous Codons ◽

Codon Composition ◽

Theoretical Predictions ◽

Highly Expressed Genes

Although the genetic code is redundant, synonymous codons for the same amino acid are not used with equal frequencies in genomes, a phenomenon termed “codon usage bias.” Previous studies have demonstrated that synonymous changes in a coding sequence can exert significantciseffects on the gene’s expression level. However, whether the codon composition of a gene can also affect the translation efficiency of other genes has not been thoroughly explored. To study how codon usage bias influences the cellular economy of translation, we massively converted abundant codons to their rare synonymous counterpart in several highly expressed genes inEscherichia coli. This perturbation reduces both the cellular fitness and the translation efficiency of genes that have high initiation rates and are naturally enriched with the manipulated codon, in agreement with theoretical predictions. Interestingly, we could alleviate the observed phenotypes by increasing the supply of the tRNA for the highly demanded codon, thus demonstrating that the codon usage of highly expressed genes was selected in evolution to maintain the efficiency of global protein translation.

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Non-AUG Translation Initiation in a Plant RNA Virus: a Forty-Amino-Acid Extension Is Added to the N Terminus of the Soil-Borne Wheat Mosaic Virus Capsid Protein

Journal of Virology ◽

10.1128/jvi.72.2.1677-1682.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 1677-1682 ◽

Cited By ~ 33

Author(s):

Yukio Shirako

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Mosaic Virus ◽

Capsid Protein ◽

Translation Initiation ◽

Deletion Mutant ◽

Rna Virus ◽

The United States ◽

Initiation Codon ◽

N Terminus

ABSTRACT RNA 2 of soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, encodes a protein previously hypothesized to be initiated at an in-frame non-AUG codon upstream of the AUG initiation codon (nucleotide positions 334 to 336) for the 19-kDa capsid protein. Site-directed mutagenesis and in vitro transcription and translation analysis indicated that CUG (nucleotides 214 to 216) is the initiation codon for a protein with a calculated molecular mass of 25 kDa composed of a 40-amino-acid extension to the N terminus of the 19-kDa capsid protein. A stable deletion mutant, which was isolated after extensive passages of a wild-type SBWMV, contained a mixture of two deleted RNA 2’s, only one of which coded for the 25-kDa protein. The amino acid sequence of the N-terminal extension was moderately conserved and the CUG initiation codon was preserved among three SBWMV isolates from Japan and the United States. This amino acid sequence conservation, as well as the retention of expression of the 25-kDa protein in the stable deletion mutant, suggests that the 25-kDa protein is functional in the life cycle of SBWMV. This is the first report of a non-AUG translation initiation in a plant RNA virus genome.

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Isolation and Amino Acid Sequence of a Protein-synthesis Inhibitor from the Seeds of Rye (Secale cereale)

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.62.1152 ◽

1998 ◽

Vol 62 (6) ◽

pp. 1152-1156 ◽

Cited By ~ 2

Author(s):

Yuji MINAMI ◽

Ken-ichi YAMAGUCHI ◽

Fumio YAGI ◽

Kenjiro TADERA ◽

Gunki FUNATSU

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Amino Acid Sequence ◽

Secale Cereale ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor

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Leucine and mTORC1: a complex relationship

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00525.2011 ◽

2012 ◽

Vol 302 (11) ◽

pp. E1329-E1342 ◽

Cited By ~ 141

Author(s):

Kayleigh M. Dodd ◽

Andrew R. Tee

Keyword(s):

Signal Transduction ◽

Protein Synthesis ◽

Amino Acid ◽

Protein Turnover ◽

Muscle Growth ◽

Protein Translation ◽

Amino Acid Availability ◽

Intracellular Amino Acid ◽

System L ◽

Mtorc1 Signaling

Amino acid availability is a rate-limiting factor in the regulation of protein synthesis. When amino acid supplies become restricted, mammalian cells employ homeostatic mechanisms to rapidly inhibit processes such as protein synthesis, which demands high levels of amino acids. Muscle cells in particular are subject to high protein turnover rates to maintain amino acid homeostasis. Mammalian target of rapamycin complex 1 (mTORC1) is an evolutionary conserved multiprotein complex that coordinates a network of signaling cascades and functions as a key mediator of protein translation, gene transcription, and autophagy. Signal transduction through mTORC1, which is centrally involved in muscle growth through enhanced protein translation, is governed by intracellular amino acid supply. The branched-chain amino acid leucine is critical for muscle growth and acts in part through activation of mTORC1. Recent research has revealed that mTORC1 signaling is coordinated primarily at the lysosomal membranes. This discovery has sparked a wealth of research in this field, revealing several different signaling molecules involved in transducing the amino acid signal to mTORC1, including the Rag GTPases, MAP4K3, and Vps34/ULK1. This review evaluates the current knowledge regarding cellular mechanisms that control and sense the intracellular amino acid pool. We discuss the role of leucine and mTORC1 in the regulation of amino acid transport via the system L and system A transporters such as LAT1 and SNAT2, as well as protein degradation via autophagic and proteasomal pathways. We also describe the complexities of energy homeostasis via AMPK and cell receptor-mediated growth signals that also converge on mTORC1. Leucine is a particularly potent regulator of protein turnover, to the extent where leucine stimulation alone is sufficient to stimulate mTORC1 signal transduction. The significance of leucine in this context is not yet known; however, recent advancements in this area will also be covered within this review.

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The Translation Initiation Factor eIF4E Is Overexpressed in Multiple Myeloma and Is Critical for Myeloma Cell Proliferation and Survival

Blood ◽

10.1182/blood.v118.21.1853.1853 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1853-1853 ◽

Cited By ~ 1

Author(s):

Shirong Li ◽

MeiHua Jin ◽

Ailing Liu ◽

Markus Y. Mapara ◽

Suzanne Lentzsch

Keyword(s):

Multiple Myeloma ◽

Protein Synthesis ◽

Translation Initiation ◽

Clinical Care ◽

Protein Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Mrna Levels ◽

Myeloma Cells ◽

Rpmi 8226

Abstract Abstract 1853 Methods: The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM. Results: By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p<0.05) proliferation by 43–62% indicating that drugs controlling protein translation inhibit MM growth. We also found that all drugs decreased expression of eIF4E dependent targets such as cyclin D1 and c-myc. Conclusion: Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

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Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

Applied and Environmental Microbiology ◽

10.1128/aem.06332-11 ◽

2012 ◽

Vol 78 (6) ◽

pp. 1724-1732 ◽

Cited By ~ 30

Author(s):

Arnau Bassegoda ◽

F. I. Javier Pastor ◽

Pilar Diaz

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Candida Antarctica ◽

Burkholderia Cenocepacia ◽

Sequence Motifs ◽

Oxyanion Hole ◽

Content Type ◽

New Family ◽

Consensus Amino Acid Sequence ◽

Four Blocks

ABSTRACTBacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genusRhodococcusand taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes fromRhodococcussp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genusRhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloningBurkholderia cenocepaciaputative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in theCandida antarcticalipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.

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