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Related Concept Videos

tRNA Activation02:26

tRNA Activation

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...
tRNA Activation02:26

tRNA Activation

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...
Tail-anchoring of Proteins in the ER Membrane01:45

Tail-anchoring of Proteins in the ER Membrane

Tail-anchored, or TA, proteins are estimated to make up to 3-5% of membrane proteins found in the eukaryotic cell. Such proteins have a single transmembrane domain located approximately 30 amino acid residues upstream from the C-terminal end. As a result, the signal recognition particle (SRP) cannot guide a TA protein to the ER membrane for cotranslational insertion. Hence, they are integrated into the ER membrane post-translationally using their C-terminal end as the anchor. TA proteins...
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.
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...
Translocation of Proteins into the Mitochondria01:19

Translocation of Proteins into the Mitochondria

Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
Cotranslational Protein Translocation01:20

Cotranslational Protein Translocation

Translocation of proteins across membranes is an ancient process that occurs even in bacteria and archaebacteria. In fact, the components of the translocation machinery are still conserved between prokaryotes and eukaryotes.
Sec61 channel partners for cotranslational translocation
During cotranslational translocation, the Sec61 channel partners with the signal recognition particle (SRP), the signal recognition particle receptor (SR), and the ribosomes to transport the nascent polypeptide chain...

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Related Experiment Video

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A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
11:08

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Published on: December 9, 2017

N-terminal protein modification using simple aminoacyl transferase substrates.

Anne M Wagner1, Mark W Fegley, John B Warner

  • 1Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, United States.

Journal of the American Chemical Society
|September 8, 2011
PubMed
Summary

This study shows that Escherichia coli aminoacyl tRNA transferase (AaT) can efficiently use simple adenosine substrates for N-terminal protein modification. This expands substrate scope and reaction scale for protein engineering while maintaining protein folding.

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Area of Science:

  • Biochemistry
  • Protein Engineering
  • Synthetic Biology

Background:

  • Escherichia coli aminoacyl tRNA transferase (AaT) modifies protein N-termini using tRNA or oligonucleotide donors.
  • Current methods for N-terminal protein modification face substrate limitations and complex synthesis.

Purpose of the Study:

  • To demonstrate AaT's ability to utilize minimal adenosine substrates for N-terminal protein modification.
  • To overcome limitations of existing protein modification techniques.

Main Methods:

  • Characterization of AaT enzymatic activity with novel aminoacyl adenosyl donors.
  • Synthesis of minimal adenosine substrates from readily available materials.
  • Assessment of reaction product inhibition on AaT activity.

Main Results:

  • AaT efficiently uses minimal adenosine substrates for N-terminal modification.
  • Adenosyl donors are synthesized in one to two steps.
  • Reaction products do not inhibit AaT activity.
  • This method avoids synthetase limitations and complex oligonucleotide synthesis.

Conclusions:

  • Adenosyl donors significantly enhance the substrate scope and reaction scale for AaT-mediated N-terminal protein modification.
  • This approach facilitates protein engineering under conditions that preserve protein folding.
  • The simplified substrate synthesis offers a more accessible method for protein functional studies.