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Related Concept Videos

tRNA Activation02:26

tRNA Activation

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Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
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Transfer RNA Synthesis02:36

Transfer RNA Synthesis

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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.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
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Improving Translational Accuracy02:07

Improving Translational Accuracy

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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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Transcription Attenuation in Prokaryotes02:42

Transcription Attenuation in Prokaryotes

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Transcriptional attenuation occurs when RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure.  Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
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Termination of Translation01:44

Termination of Translation

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The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
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Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

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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.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
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Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Han-Kai Jiang1,2,3,4, Nicole L Ambrose1, Christina Z Chung1

  • 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.

Proceedings of the National Academy of Sciences of the United States of America
|February 14, 2023
PubMed
Summary

Scientists engineered split orthogonal aminoacyl-tRNA synthetases (o-aaRS) to control gene translation. These synthetic biology tools act as AND gates, enabling precise gene expression regulation and biosensing in bacteria and human cells.

Keywords:
genetic code expansionnoncanonical amino acidspyrrolysyl-tRNA synthetasestop codon suppressionsynthetic biology

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Gene expression regulation is crucial for biotechnology and therapeutics.
  • Existing tools primarily target transcriptional control, with limited options for translational regulation.

Purpose of the Study:

  • To engineer novel synthetic biology tools for controlling gene translation.
  • To develop split orthogonal aminoacyl-tRNA synthetases (o-aaRS) for precise gene expression modulation.

Main Methods:

  • Designed and engineered split o-aaRS using chemically induced dimerization domains.
  • Utilized small molecules (rapamycin, abscisic acid) to control o-aaRS activity.
  • Demonstrated function in *Escherichia coli* and human cells.

Main Results:

  • Split o-aaRS conditionally suppress stop codons in response to specific molecular inputs.
  • These systems function as genetically encoded AND gates, requiring dual molecular signals for activation.
  • Validated split o-aaRS as biosensors for detecting protein-protein interactions relevant to cancer and viral infections (e.g., SARS-CoV-2).

Conclusions:

  • Split o-aaRS provide a novel platform for precise translational control of gene expression.
  • The developed tools offer versatile applications in synthetic biology, biosensing, and therapeutic development.