tRNA concentration fine tunes protein solubility

Fedyunin, Ivan; Lehnhardt, Lothar; Böhmer, Nadine; Kaufmann, Paul; Zhang, Gong; Ignatova, Zoya

doi:10.1016/j.febslet.2012.07.012

Cited by 66 publications

(58 citation statements)

References 35 publications

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“…Codon usage has a substantive effect on co-translational protein folding by altering the rate at which the nascent polypeptide is synthesized [47]. Rare, or slow, codons are present in specific regions of mRNA messages and function to alter translation rates, and thus protein folding, in mRNA segments that correspond to protein domain boundaries [48], [49] or in the transition from unstructured to structured regions of the nascent polypeptide chain [50], allowing structural elements additional time to fold.…”

Section: Discussionmentioning

confidence: 99%

“…Attempts to manipulate the expression of heterologous proteins by mitigating the effects of rare codons, through the use of synthetic genes or accessory plasmids encoding tRNA molecules for rare codons, have had mixed results. The increased translation rates associated with these strategies appear to uncouple the co-translational process of chain elongation and protein folding leading to high amounts of misfolded, insoluble protein [47], [51].…”

Section: Discussionmentioning

confidence: 99%

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Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

et al. 2013

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The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

et al. 2013

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“…Further evidence for an important role of nonoptimal, more slowly translated codons stems from findings that overexpression of tRNAs corresponding to nonoptimal codons disrupts protein homeostasis and leads to widespread aggregation 56 . Our analysis found clusters of nonoptimal codons leading to a region of three to seven codons that is translated at significantly lower speed.…”

Section: Methodsmentioning

confidence: 99%

Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo

Pechmann

Chartron

Frydman

2014

Nat Struct Mol Biol

208

195

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The genetic code allows most amino acids a choice of optimal and nonoptimal codons. We report that synonymous codon choice is tuned to promote interaction of nascent polypeptides with the signal recognition particle (SRP), which assists in protein translocation across membranes. Cotranslational recognition by the SRP in vivo is enhanced when mRNAs contain nonoptimal codon clusters 35–40 codons downstream of the SRP-binding site, the distance that spans the ribosomal polypeptide exit tunnel. A local translation slowdown upon ribosomal exit of SRP-binding elements in mRNAs containing these nonoptimal codon clusters is supported experimentally by ribosome profiling analyses in yeast. Modulation of local elongation rates through codon choice appears to kinetically enhance recognition by ribosome-associated factors. We propose that cotranslational regulation of nascent-chain fate may be a general constraint shaping codon usage in the genome.

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“…Increasing cellular tRNA levels is expected to raise the translation rate of codons with low levels of cognate tRNA, reducing translational pauses. Increasing tRNA levels in E. coli produced an overall decrease in soluble protein and increase in insoluble aggregates, suggesting translational pauses are important for the folding of many proteins (43).…”

Section: Cotranslational Protein Foldingmentioning

confidence: 99%

Roles for Synonymous Codon Usage in Protein Biogenesis

Chaney¹,

Clark

2015

Annu. Rev. Biophys.

264

255

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Owing to the degeneracy of the genetic code, a protein sequence can be encoded by many different synonymous mRNA coding sequences. Synonymous codon usage was once thought to be functionally neutral, but evidence now indicates it is shaped by evolutionary selection and affects other aspects of protein biogenesis beyond specifying the amino acid sequence of the protein. Synonymous rare codons, once thought to have only negative impacts on the speed and accuracy of translation, are now known to play an important role in diverse functions, including regulation of cotranslational folding, covalent modifications, secretion, and expression level. Mutations altering synonymous codon usage are linked to human diseases. However, much remains unknown about the molecular mechanisms connecting synonymous codon usage to efficient protein biogenesis and proper cell physiology. Here we review recent literature on the functional effects of codon usage, including bioinformatics approaches aimed at identifying general roles for synonymous codon usage.

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tRNA concentration fine tunes protein solubility

Cited by 66 publications

References 35 publications

Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo

Roles for Synonymous Codon Usage in Protein Biogenesis

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