Optimization of Translation Profiles Enhances Protein Expression and Solubility

mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

[1]  Pamela A. Silver,et al.  Coupling and coordination in gene expression processes: a systems biology view , 2008, Nature Reviews Genetics.

[2]  N. Sonenberg,et al.  Principles of translational control: an overview. , 2012, Cold Spring Harbor perspectives in biology.

[3]  X. Xia.  How optimized is the translational machinery in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae? , 1998, Genetics.

[4]  Manolis Kellis,et al.  Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo , 2013, Nature.

[5]  M. Carmen Romano,et al.  Ribosome Traffic on mRNAs Maps to Gene Ontology: Genome-wide Quantification of Translation Initiation Rates and Polysome Size Regulation , 2013, PLoS Comput. Biol..

[6]  C. Dobson.  Protein folding and misfolding , 2003, Nature.

[7]  Peter J McCormick,et al.  Nascent Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the Ribosome and in Their Exposure to Ribosomal Proteins , 2004, Cell.

[8]  Jian-Rong Yang,et al.  Codon-by-Codon Modulation of Translational Speed and Accuracy Via mRNA Folding , 2014, PLoS biology.

[9]  Germán L. Rosano,et al.  Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain , 2009, Microbial cell factories.

[10]  Tamir Tuller,et al.  Codon bias, tRNA pools, and horizontal gene transfer , 2011, Mobile genetic elements.

[11]  A. Hinnebusch,et al.  Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets , 2009, Cell.

[12]  E. O’Shea,et al.  An Integrated Approach Reveals Regulatory Controls on Bacterial Translation Elongation , 2014, Cell.

[13]  Jelena Repar,et al.  Translational Selection Is Ubiquitous in Prokaryotes , 2010, PLoS genetics.

[14]  I. Stansfield,et al.  Ribosome recycling induces optimal translation rate at low ribosomal availability , 2014, Journal of The Royal Society Interface.

[15]  David L. Wheeler,et al.  GenBank , 2015, Nucleic Acids Res..

[16]  Z. Ignatova,et al.  tRNA concentration fine tunes protein solubility , 2012, FEBS letters.

[17]  A. Komar,et al.  A pause for thought along the co-translational folding pathway. , 2009, Trends in biochemical sciences.

[18]  Reinhard Wolf,et al.  Coding-Sequence Determinants of Gene Expression in Escherichia coli , 2009 .

[19]  Z. Ignatova,et al.  Systematic identification of tRNAome and its dynamics in Lactococcus lactis , 2014, Molecular microbiology.

[20]  M. Turner.  Is transcription the dominant force during dynamic changes in gene expression? , 2011, Advances in experimental medicine and biology.

[21]  Hendrik J. Viljoen,et al.  Ribosome kinetics and aa-tRNA competition determine rate and fidelity of peptide synthesis , 2007, Comput. Biol. Chem..

[22]  Jürgen Pleiss,et al.  The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions , 2004, Bioinform..

[23]  F. Oesch,et al.  Epoxide hydrolases: structure, function, mechanism, and assay. , 2005, Methods in enzymology.

[24]  C. Kurland,et al.  Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. , 1996, Journal of molecular biology.

[25]  Liisa Holm,et al.  Dali server: conservation mapping in 3D , 2010, Nucleic Acids Res..

[26]  S. Capaccioli,et al.  Post‐transcriptional regulation of gene expression by degradation of messenger RNAs , 2003, Journal of cellular physiology.

[27]  S. Govindarajan,et al.  Codon bias and heterologous protein expression. , 2004, Trends in biotechnology.

[28]  Burkhard Rost,et al.  The PredictProtein server , 2003, Nucleic Acids Res..

[29]  Koreaki Ito,et al.  Divergent stalling sequences sense and control cellular physiology. , 2010, Biochemical and biophysical research communications.

[30]  I. Stansfield,et al.  Halting a cellular production line: responses to ribosomal pausing during translation , 2007, Biology of the cell.

[31]  Tao Pan,et al.  Tissue-Specific Differences in Human Transfer RNA Expression , 2006, PLoS genetics.

[32]  P. Spencer,et al.  Silent substitutions predictably alter translation elongation rates and protein folding efficiencies. , 2012, Journal of molecular biology.

[33]  Peter F. Stadler,et al.  ViennaRNA Package 2.0 , 2011, Algorithms for Molecular Biology.

[34]  D. Janssen,et al.  Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium radiobacter AD1* , 1997, The Journal of Biological Chemistry.

[35]  Samuel Wagner,et al.  Tuning Escherichia coli for membrane protein overexpression , 2008, Proceedings of the National Academy of Sciences.

[36]  J. Elf,et al.  Selective Charging of tRNA Isoacceptors Explains Patterns of Codon Usage , 2003, Science.

[37]  Sriram Kosuri,et al.  Causes and Effects of N-Terminal Codon Bias in Bacterial Genes , 2013, Science.

[38]  Randall L. Kincaid,et al.  Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host , 2008, PloS one.

[39]  B. Freeman,et al.  Slowing bacterial translation speed enhances eukaryotic protein folding efficiency. , 2010, Journal of molecular biology.

[40]  Jimin Pei,et al.  PROMALS: towards accurate multiple sequence alignments of distantly related proteins , 2007, Bioinform..

[41]  Fran Supek,et al.  On Relevance of Codon Usage to Expression of Synthetic and Natural Genes in Escherichia coli , 2010, Genetics.

[42]  Sebastian M. Waszak,et al.  A Dual Program for Translation Regulation in Cellular Proliferation and Differentiation , 2014, Cell.

[43]  Zoya Ignatova,et al.  Transient ribosomal attenuation coordinates protein synthesis and co-translational folding , 2009, Nature Structural &Molecular Biology.

[44]  J. Kappes,et al.  A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein* , 2010, The Journal of Biological Chemistry.

[45]  Mark Gerstein,et al.  Revisiting the codon adaptation index from a whole-genome perspective: analyzing the relationship between gene expression and codon occurrence in yeast using a variety of models. , 2003, Nucleic acids research.

[46]  N. Blüthgen,et al.  Molecular Systems Biology 9; Article number 675; doi:10.1038/msb.2013.32 Citation: Molecular Systems Biology 9:675 , 2022 .

[47]  B. Mueller‐Roeber,et al.  Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation , 2010, Nucleic acids research.

[48]  J. Belasco.  All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay , 2010, Nature Reviews Molecular Cell Biology.

[49]  M Carmen Romano,et al.  Controlling translation elongation efficiency: tRNA regulation of ribosome flux on the mRNA. , 2014, Biochemical Society transactions.

[50]  Alan Villalobos,et al.  Design Parameters to Control Synthetic Gene Expression in Escherichia coli , 2009, PloS one.

[51]  Jürgen Eck,et al.  Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space. , 2002, Current opinion in biotechnology.

[52]  Zoya Ignatova,et al.  Folding at the birth of the nascent chain: coordinating translation with co-translational folding. , 2011, Current opinion in structural biology.

[53]  Zoya Ignatova,et al.  Generic Algorithm to Predict the Speed of Translational Elongation: Implications for Protein Biogenesis , 2009, PloS one.

[54]  S. Kanaya,et al.  Studies of codon usage and tRNA genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene expression level and species-specific diversity of codon usage based on multivariate analysis. , 1999, Gene.

[55]  T. Pan,et al.  A Role for tRNA Modifications in Genome Structure and Codon Usage , 2012, Cell.

[56]  Antonis Rokas,et al.  Non-optimal codon usage is a mechanism to achieve circadian clock conditionality , 2013, Nature.

[57]  A. Komar,et al.  Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation , 1999, FEBS letters.

[58]  D. Drummond,et al.  A Nutrient-Driven tRNA Modification Alters Translational Fidelity and Genome-wide Protein Coding across an Animal Genus , 2014, PLoS biology.

[59]  Jürgen Pleiss,et al.  Sequence and structure of epoxide hydrolases: A systematic analysis , 2004, Proteins.