Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

Abstract

Post-translational modifications (PTMs) endow proteins with new properties to respond to environmental changes or growth needs. With the development of advanced proteomics techniques, hundreds of distinct types of PTMs have been observed in a wide range of proteins from bacteria, archaea, and eukarya. To identify the roles of these PTMs, scientists have applied various approaches. However, high dynamics, low stoichiometry, and crosstalk between PTMs make it almost impossible to obtain homogeneously modified proteins for characterization of the site-specific effect of individual PTM on target proteins. To solve this problem, the genetic code expansion (GCE) strategy has been introduced into the field of PTM studies. Instead of modifying proteins after translation, GCE incorporates modified amino acids into proteins during translation, thus generating site-specifically modified proteins at target positions. In this review, we summarize the development of GCE systems for orthogonal translation for site-specific installation of PTMs.

Reading and Writing the Ubiquitin Code Using Genetic Code Expansion

“…Chin and colleagues originally reported an efficient Nɛ-acetyllysyl-tRNA synthetase/tRNA orthogonal pair with high translational fidelity towards the amber stop codon. [76] Selection of the MbRS with randomized positions at Leu266, Leu270, Tyr271, Leu274, Cys313, and Trp383 led to two predominant mutants that selectively encode Nɛ-actyllysine (Figure 8A,25). These mutant synthetases AcKRS-1 (L270I, Y271F, L274A, C313F and D76G) and AcKRS-2 (L270I, Y271L, L274A, and C313F) were able to discriminate the minimal acetyl moiety for suppression of the stop codon.…”

“…Their uses span everywhere from biorthogonal labeling in live cells to expanded and enhanced chemistries for biocatalysis. [22,23] A powerful application of this technology lies in its ability to produce homogenously modified proteins bearing naturally occurring PTMs, [24][25][26] a feature that has been recently exploited to generate and study the many different Ub proteoforms and expanded chemistries. In this review, we discuss recent advances on the use of genetic code expansion to study how the ubiquitin code is 'written', 'read', and 'erased'.…”