Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte

Abstract

Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA (pylT) from Methanosarcina barkeri fusaro is orthogonal and highly competent for genetic code expansion experiments in the Xenopus oocyte. The data show that pylT is amendable to chemical acylation in vitro; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the pylT is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus, pylT is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the Xenopus oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.

Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape

“…2 B, we have quantified the amount of ionic current observed at +50 mV at each site, in comparison with the same cRNA co-injected with unacylated, full-length (pCA-ligated) pyrrolysine tRNA. Currents were not seen in the absence of appended amino acid, reflective of the orthogonality of this tRNA species in the X. laevis oocyte (Infield et al, 2018). Ionic currents were not observed from injection of citrulline-acylated tRNA with cRNA encoding R2TAG (R365) or R6TAG (R377), signifying either that these sites were not efficiently “rescued” by Pyl-citrulline or that citrulline incorporation rendered the channels nonfunctional.…”

Therapeutic promise of engineered nonsense suppressor tRNAs

“…With the steady development of GCE, first achieved in E. coli (Furter, 1998; L. Wang, Czaplinski, et al, 2001), its successful implementation has been realized in a number of cell types and organisms, including yeast (Hancock et al, 2010; Q. Wang & Wang, 2008), eukaryotic cell culture (Elsasser et al, 2016; Gautier et al, 2010; Hino et al, 2005; Mukai et al, 2008; Sakamoto et al, 2002; Xiao et al, 2013), C. elegans (Greiss & Chin, 2011; Parrish et al, 2012), Drosophila (Bianco et al, 2012; Elliott et al, 2014), zebrafish (Brown & Deiters, 2019; Chen et al, 2017; Liu et al, 2017), and mouse brain (Ernst et al, 2016; Kang et al, 2013; Maywood et al, 2018; Zheng et al, 2017). To implement GCE in these cells and organisms, the o‐tRNA was delivered in several different fashions including transient transfection (Hino et al, 2005; Sakamoto et al, 2002; Schmied et al, 2014; Xiao et al, 2013), viral transduction (Chatterjee et al, 2013; Shen et al, 2011; Zheng et al, 2017), genomic integration (Elsasser et al, 2016; Sakamoto et al, 2002), or injected as RNA (Infield et al, 2018; Noren et al, 1989; Saks et al, 1996). GCE methods have demonstrated that o‐tRNAs can be delivered with different methods as either DNA or RNA, and are stable, heritable, and tolerable in a variety of eukaryotic cells, zebrafish, and mice (Chen et al, 2017; Elsasser et al, 2016).…”

Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor

“…Both the Tetrahymena thermophila (THG73) 54 and Pyrrolysine (Pyl, from Barkeri fusaro ) 55 suppressor tRNAs were used in these studies. We tested THG73- and Pyl-tRNAs at all positions and for 5 of the 8 positions investigated, no difference was observed with either of these suppressor tRNAs.…”