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Related Concept Videos

Improving Translational Accuracy02:07

Improving Translational Accuracy

Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
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Nonsense-mediated mRNA Decay02:27

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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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Transfer RNA Synthesis02:36

Transfer RNA Synthesis

One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems
06:18

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Published on: April 26, 2019

Transfer RNA misidentification scrambles sense codon recoding.

Radha Krishnakumar1, Laure Prat, Hans-Rudolf Aerni

  • 1Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850 (USA).

Chembiochem : a European Journal of Chemical Biology
|September 4, 2013
PubMed
Summary
This summary is machine-generated.

Genetic code expansion aims to incorporate new amino acids. Researchers attempted to reassign the CGG codon in Mycoplasma capricolum, but found it still translated to arginine.

Keywords:
aminoacyl-tRNA synthetase recognitionanticodonsgene technologysense codon recodingtRNA

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Genetics

Background:

  • Sense codon recoding enables genetic code expansion.
  • This requires an unused codon and an orthogonal tRNA synthetase:tRNA pair.
  • The Mycoplasma capricolum genome has limited CGG arginine codons without a dedicated tRNA(Arg).

Purpose of the Study:

  • To reassign the CGG codon to pyrrolysine in Mycoplasma capricolum.
  • To investigate the recognition of a synthetic tRNA by endogenous synthetases.
  • To assess the feasibility of sense codon recoding for genetic code expansion.

Main Methods:

  • Engineered M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA(Pyl)(CCG), and pyrrolysine biosynthesis genes.
  • Utilized mass spectrometry to analyze codon translation.
  • Investigated tRNA recognition by endogenous arginyl-tRNA synthetase.

Main Results:

  • The synthetic tRNA(Pyl)(CCG) was efficiently recognized by the endogenous arginyl-tRNA synthetase.
  • Mass spectrometry confirmed that CGG codons were translated as arginine in the presence of tRNA(Pyl)(CCG).
  • This indicates tRNA misidentification by endogenous aminoacyl-tRNA synthetases.

Conclusions:

  • Sense codon recoding for genetic code expansion faces challenges with endogenous tRNA synthetase recognition.
  • Overcoming tRNA misidentification is crucial for successful genetic code expansion.
  • The study highlights the complexity of engineering orthogonal translation systems.