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Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

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Published on: December 26, 2020

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons.

Alexandre Ambrogelly¹, Sarath Gundllapalli, Stephanie Herring

¹Department of Molecular Biophysics and Biochemistry and Chemistry, Yale University, New Haven, CT 06520-8114, USA.

Proceedings of the National Academy of Sciences of the United States of America

|March 16, 2007

Summary

Pyrrolysine (Pyl), the 22nd amino acid, is attached to its tRNA by pyrrolysyl-tRNA synthetase (PylRS). Researchers identified key RNA elements in tRNA(Pyl) essential for this recognition and aminoacylation process.

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Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

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Published on: March 28, 2016

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Pyrrolysine (Pyl) is the 22nd naturally encoded amino acid.
Pyrrolysine is acylated to its specific UAG suppressor tRNA(Pyl) by pyrrolysyl-tRNA synthetase (PylRS).

Purpose of the Study:

To determine the essential RNA elements for tRNA(Pyl) recognition and aminoacylation by PylRS in vivo.
To investigate the potential of tRNA(Pyl) variants for decoding multiple codons.

Main Methods:

Testing 42 Methanosarcina barkeri tRNA(Pyl) variants in Escherichia coli for lac amber mutation suppression.
Utilizing a yeast three-hybrid system to assess in vivo binding of tRNA(Pyl) mutants to PylRS.
Measuring in vitro tRNA(Pyl) aminoacylation.
Transplanting tRNA(Pyl) identity elements into a bovine tRNA(Ser) scaffold.

Main Results:

Identified key tRNA(Pyl) identity elements: discriminator base, acceptor stem first base pair, T-stem base pair G51:C63, and anticodon flanking nucleotides U33 and A37.
Chimeric tRNAs with transplanted identity elements were active both in vitro and in vivo.
A tRNA(Pyl) variant efficiently suppressed a lac opal U4 mutation, as the anticodon is not crucial for PylRS recognition.

Conclusions:

tRNA(Pyl) identity elements are critical for specific aminoacylation by PylRS.
The tRNA(Pyl):PylRS pair is highly orthogonal, facilitating the incorporation of pyrrolysine into the genetic code.
Engineered tRNA(Pyl) variants show potential for decoding diverse codons, expanding the genetic code.