G/U and certain wobble position mismatches as possible main causes of amino acid misincorporations.

A mass spectrometry-based method was developed to measure amino acid substitutions directly in proteins down to a level of 0.001%. When applied to recombinant proteins expressed in Escherichia coli, monoclonal antibodies expressed in mammalian cells, and human serum albumin purified from three human subjects, the method revealed a large number of amino acid misincorporations at levels of 0.001-0.1%. The detected misincorporations were not random but involved a single-base difference between the codons of the corresponding amino acids. The most frequent base differences included a change from G to A, corresponding to a G(mRNA)/U(tRNA) base pair mismatch during translation. We concluded that under balanced nutrients, G(mRNA)/U(tRNA) mismatches at any of the three codon positions and certain additional wobble position mismatches (C/U and/or U/U) are the main causes of amino acid misincorporations. The hypothesis was tested experimentally by monitoring the levels of misincorporation at several amino acid sites encoded by different codons, when a protein with the same amino acid sequence was expressed in E. coli using 13 different DNA sequences. The observed levels of misincorporation were different for different codons and agreed with the predicted levels. Other less frequent misincorporations may occur due to G(DNA)/U(mRNA) mismatch during transcription, mRNA editing, U(mRNA)/G(tRNA) mismatch during translation, and tRNA mischarging.

[1]  D. Michels,et al.  Identification of codon-specific serine to asparagine mistranslation in recombinant monoclonal antibodies by high-resolution mass spectrometry. , 2009, Analytical chemistry.

[2]  M. Rodnina,et al.  Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms. , 2001, Annual review of biochemistry.

[3]  R. Pease,et al.  A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine , 1987, Cell.

[4]  D. Turner,et al.  Free energy contributions of G.U and other terminal mismatches to helix stability. , 1986, Biochemistry.

[5]  P. Doty,et al.  Complementary Oligonucleotide Binding to the Anticodon Loop of fMet-transfer RNA , 1970, Nature.

[6]  C. Kurland,et al.  Codon‐specific missense errors in vivo. , 1983, The EMBO journal.

[7]  J. Friesen,et al.  “Two out of three” codon reading leading to mistranslation in vivo , 1980, Molecular and General Genetics MGG.

[8]  W. Fitch,et al.  The biological significance of G-T/G-U mispairing in nucleic acid secondary structure. , 1985, Journal of theoretical biology.

[9]  C. Francklyn.  DNA polymerases and aminoacyl-tRNA synthetases: shared mechanisms for ensuring the fidelity of gene expression. , 2008, Biochemistry.

[10]  C. Wilke,et al.  The evolutionary consequences of erroneous protein synthesis , 2009, Nature Reviews Genetics.

[11]  M. Ibba,et al.  Cellular mechanisms that control mistranslation , 2010, Nature Reviews Microbiology.

[12]  Zhongqi Zhang,et al.  Large-scale identification and quantification of covalent modifications in therapeutic proteins. , 2009, Analytical chemistry.

[13]  T. Oas,et al.  High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli. , 1996, Journal of molecular biology.

[14]  Zhongqi Zhang,et al.  Targeted codon optimization improves translational fidelity for an Fc fusion protein , 2012, Biotechnology and bioengineering.

[15]  D. Turner,et al.  Energetics of internal GU mismatches in ribooligonucleotide helixes. , 1986, Biochemistry.

[16]  Hani S. Zaher,et al.  Fidelity at the Molecular Level: Lessons from Protein Synthesis , 2009, Cell.

[17]  Zhongqi Zhang.  Prediction of low-energy collision-induced dissociation spectra of peptides. , 2004, Analytical chemistry.

[18]  Z W Gu,et al.  Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon. , 1987, Science.

[19]  Liang-Yu Shih,et al.  An improved trypsin digestion method minimizes digestion-induced modifications on proteins. , 2009, Analytical biochemistry.

[20]  Zhongqi Zhang,et al.  Rearrangement of terminal amino acid residues in peptides by protease-catalyzed intramolecular transpeptidation. , 2006, Analytical biochemistry.

[21]  P. Farabaugh,et al.  The frequency of translational misreading errors in E. coli is largely determined by tRNA competition. , 2006, RNA.

[22]  Kai A Reidegeld,et al.  Tryptic transpeptidation products observed in proteome analysis by liquid chromatography‐tandem mass spectrometry , 2005, Proteomics.

[23]  V. Ramakrishnan,et al.  First published online as a Review in Advance on February 25, 2005 STRUCTURAL INSIGHTS INTO TRANSLATIONAL , 2022 .

[24]  Yao-ming Huang,et al.  Discovery and Investigation of Misincorporation of Serine at Asparagine Positions in Recombinant Proteins Expressed in Chinese Hamster Ovary Cells , 2009, The Journal of Biological Chemistry.

[25]  S. Limmer.  Mismatch base pairs in RNA. , 1997, Progress in nucleic acid research and molecular biology.

[26]  Zhongqi Zhang.  Automated Precursor Ion Exclusion During LC-MS/MS Data Acquisition for Optimal Ion Identification , 2012, Journal of The American Society for Mass Spectrometry.

[27]  Mingyao Li,et al.  Widespread RNA and DNA Sequence Differences in the Human Transcriptome , 2011, Science.

[28]  G. Varani,et al.  The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems. , 2000, EMBO reports.

[29]  J. Gallant,et al.  An estimate of the global error frequency in translation , 1982, Molecular and General Genetics MGG.

[30]  Brenda L Bass,et al.  RNA editing by adenosine deaminases that act on RNA. , 2002, Annual review of biochemistry.

[31]  M. Ibba,et al.  Aminoacyl-tRNA synthesis and translational quality control. , 2009, Annual review of microbiology.

[32]  Zhongqi Zhang,et al.  Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. , 2005, Analytical chemistry.

[33]  Paul F Agris,et al.  tRNA's wobble decoding of the genome: 40 years of modification. , 2007, Journal of molecular biology.

[34]  N. Navaratnam,et al.  RNA editing: cytidine to uridine conversion in apolipoprotein B mRNA. , 2000, Biochimica et biophysica acta.

[35]  P. Masters,et al.  High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae al homeodomain expressed in Escherichia coli , 1998, Protein science : a publication of the Protein Society.

[36]  V. Ramakrishnan,et al.  Selection of tRNA by the Ribosome Requires a Transition from an Open to a Closed Form , 2002, Cell.

[37]  J. Gallant,et al.  Mistranslation in E. coli , 1977, Cell.

[38]  G. Church,et al.  Genome-Wide Identification of Human RNA Editing Sites by Parallel DNA Capturing and Sequencing , 2009, Science.

[39]  Zhongqi Zhang.  Prediction of collision-induced-dissociation spectra of peptides with post-translational or process-induced modifications. , 2011, Analytical chemistry.

[40]  P. Schimmel,et al.  Evidence for a unique first position codon-anticodon mismatch in vivo. , 1988, Journal of molecular biology.

[41]  Paul Chen,et al.  Control of misincorporation of serine for asparagine during antibody production using CHO cells , 2010, Biotechnology and bioengineering.

[42]  G. Buxton,et al.  Critical Review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (⋅OH/⋅O− in Aqueous Solution , 1988 .

[43]  M. Tomita,et al.  Sensitized photooxidation of histidine and its derivatives. Products and mechanism of the reaction. , 1969, Biochemistry.

[44]  M. Huddleston,et al.  Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli. , 2003, Protein expression and purification.

[45]  Zhongqi Zhang,et al.  Prediction of collision-induced dissociation spectra of common N-glycopeptides for glycoform identification. , 2010, Analytical chemistry.

[46]  Zhongqi Zhang,et al.  Retention Time Alignment of LC/MS Data by a Divide-and-Conquer Algorithm , 2012, Journal of The American Society for Mass Spectrometry.

[47]  J. Pollard,et al.  Stuttering: high-level mistranslation in animal and bacterial cells. , 1978, Proceedings of the National Academy of Sciences of the United States of America.