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Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation
- Source :
- Nucleic Acids Research
- Publication Year :
- 2021
- Publisher :
- Oxford University Press (OUP), 2021.
-
Abstract
- In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (MeAAs), is less efficient, especially when MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of MeAAs, achieving the incorporation of nine distinct MeAAs into both linear and thioether-macrocyclic peptide scaffolds.
- Subjects :
- AcademicSubjects/SCI00010
Stereochemistry
Oligonucleotides
NAR Breakthrough Article
Peptide
Peptide Elongation Factor Tu
RNA, Transfer, Amino Acyl
Biology
010402 general chemistry
Methylation
01 natural sciences
Ribosome
03 medical and health sciences
Escherichia coli
Genetics
Amino Acids
Thermus
Base Pairing
Peptide sequence
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
Binding Sites
Base Sequence
Translation (biology)
0104 chemical sciences
Amino acid
Elongation factor
Kinetics
chemistry
Protein Biosynthesis
Transfer RNA
Nucleic Acid Conformation
Thermodynamics
Peptidomimetics
Genetic Engineering
EF-Tu
Protein Binding
Subjects
Details
- ISSN :
- 13624962 and 03051048
- Volume :
- 49
- Database :
- OpenAIRE
- Journal :
- Nucleic Acids Research
- Accession number :
- edsair.doi.dedup.....e3e5e729152068c763c34c49d6f87dca