Genetic code expansion as a tool to study regulatory processes of transcription

Abstract

The expansion of the genetic code with non-canonical amino acids (ncAA) enables the chemical and biophysical properties of proteins to be tailored, inside cells, with a previously unattainable level of precision. A wide range of ncAA with functions not found in canonical amino acids have been genetically encoded in recent years and have delivered insights into biological processes that would be difficult to access with traditional approaches of molecular biology. A major field for the development and application of novel ncAA-functions has been transcription and its regulation. This is particularly attractive, since advanced DNA sequencing- and proteomics-techniques continue to deliver vast information on these processes on a global level, but complementing methodologies to study them on a detailed, molecular level and in living cells have been comparably scarce. In a growing number of studies, genetic code expansion has now been applied to precisely control the chemical properties of transcription factors, RNA polymerases and histones, and this has enabled new insights into their interactions, conformational changes, cellular localizations and the functional roles of posttranslational modifications.

Keywords: epigenetics; genetic code expansion; non-canonical amino acids; nucleic acid interactions; protein; transcription.

Figure 1

Overview of the opportunities that have been opened by the use of genetic code expansion in the studying of various aspects of transcription and its regulation. DNA is shown in gray, RNA in cyan.

Scheme 1

Structures of non-canonical amino acids (ncAA) used in the reviewed studies. ¹: for inorporation of ncAA 6, different Nε-protected precursors were genetically encoded followed by posttranslational deprotection in vitro (see also Scheme 2A). ²: for incorporation of ncAA 7, Nε-Boc-L-lysine was genetically encoded and dimethylation was achieved posttranslationally after deprotection in vitro (see also (Scheme 2B). ³: ncAA 10–13 were incorporated into proteins by incorporation of selenocysteine-derivatives, oxidative elimination to dehydroalanine 20 and subsequent michael additions with thiols (see also (Scheme 2C).

Figure 2

Photocrosslinking studies with an expanded genetic code. (A) Interaction of the prototypical transcriptional activator VP16 with the nucleosome remodeling complex Swi/Snf. (B) Model showing part of the central cleft of RNA polymerase II in the transcription preinitiation complex bound to the general transcrption factors TFIIE and the Ssl2 subunit of TFIIH. Three winged helix (WH) domains of TFIIE are shown in blue, magenta and dark brown, the Ssl2 subunit in light brown. RNA polymerase II is shown in gray, amino acids analyzed in photocrosslinking studies in orange. Adapted by permission from Macmillan Publishers Ltd: Nature Structural and Molecular Biology (Grunberg et al., 2012), copyright 2012. (C) Red-light controlled Protein-RNA crosslinking using ncAA 2 bearing a furan moiety. Top: activation of the furan by oxidation with singlet oxygen, resulting in a γ-keto-enal. Middle: Proposed mechanism for the formation of a cyclic adduct between the γ-keto-enal and cytosine. Lower part: Left: HIV-1 TAR. Right: Arginine-rich motif of HIV-1 TAT and incorporation positions of furan-bearing ncAA 2.

Figure 3

Photoactivation of T7 RNA polymerase using photocaged lysine 5. (A) General principle of activation of transcription of an anti-Eg5 shRNA. (B) Position of lysine used for replacement with 5, resulting in an inactive T7 RNA polymerase. (C) Photoactivation of trancription of anti-Eg5 shRNA and subsequent RNA_i -knockout of Eg5, resulting in a binuclear phenotype. Adapted with permission from Hemphill et al. (2013). Copyright (2013) American Chemical Society.

Scheme 2

Synthetic strategies for the introduction of different lysine PTM via genetic code expansion. (A) Introduction of Nε-methyl-L-lysine. (B) Introduction of Nε-, Nε-dimethyl-L-lysine. (C) Flexible introduction of various PTM in form of L-lysine mimicks.