470105 Exploiting Structural Effects of Post-Transcriptional RNA Modifications to Engineer Orthogonality of Amber Suppressor Transfer RNAs for Genetic Code Expansion

Monday, November 14, 2016: 9:48 AM
Continental 8 (Hilton San Francisco Union Square)
Kevin C. Baldridge1, Andre C. Maranhao2, Emmalie Berkovsky1, Anirudh Sivakumar1, Andrew D. Ellington2 and Lydia M. Contreras1, (1)McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, (2)Center for Systems & Synthetic Biology, University of Texas at Austin, Austin, TX

The ability to introduce non-canonical amino acids through expansion of the genetic code is also of great interest for producing proteins with new functionalities1. To this end, suppressor tRNAs (tRNAs that suppress stop codons by decoding them for a 21st amino acid), are often taken from other organisms with their corresponding synthetases and transplanted into a more conventional production organism such as E. coli. These suppressor tRNAs often reduce cellular fitness due to mischarging by host synthetases or inaccuracies in decoding such as frameshifting2. As such, approaches for improving the orthogonality (not being charged by host aminoacyl-tRNA synthetases) of suppressor tRNAs are critical for effective expansion of the genetic code. One striking feature of tRNAs is their abundance of post-transcriptionally modifications (PTMs), which are generally believed to play pivotal roles in fine tuning the structural flexibility of RNAs to maintain robust functions even under non-ideal conditions3,4. Furthermore, many positions known to be modified in E. coli tRNAs are tightly linked to determinants of identity for various tRNA isotypes5,6, highlighting the potential for taking advantage of PTMs for improving orthogonality of engineered suppressor tRNAs.

The main premise of this work is that strategic engineering locations to rationally introduce tRNA orthogonality can be identified an exploited by a combination of conventional genetics and local intracellular structural accessibility probing methods. This strategy is of impact as inferences about PTMs are difficult to make in vivo. Specifically, we have used a modified version of a tool developed in our lab for studying RNA structural accessibility in living cells7 to characterize the structural flexibility for various domains in native E. coli tRNAs as well as in several engineered Methanocaldococcus jannaschii suppressor tRNAs. The changes in flexibility associated with various PTMs were then characterized using genetic strain that were presumed to lack the PTMs of interest (given deletions of the corresponding enzyme (s) responsible for these modifications). After correlating the orthogonality and mischarging frequency with changes in PTM-dependent structural flexibility, this work highlights the importance of PTMs in modulating molecular flexibility as a key characteristic for engineering strong suppressor tRNAs. As such, this project demonstrates the value of considering RNA post-transcriptional modifications in the design of orthogonal systems for applications in synthetic biology.

  1. Young, T. S. & Schultz, P. G. Beyond the canonical 20 amino acids: expanding the genetic lexicon. J. Biol. Chem. 285, 11039–44 (2010).
  2. Kurland, C. G. Translational accuracy and the fitness of bacteria. Annu. Rev. Genet. 26, 29–50 (1992).
  3. Jackman, J. E. & Alfonzo, J. D. Transfer RNA modifications: nature’s combinatorial chemistry playground. Wiley Interdiscip. Rev. RNA 4, 35–48 (2013).
  4. Baldridge, K. C. & Contreras, L. M. Functional implications of ribosomal RNA methylation in response to environmental stress. Crit. Rev. Biochem. Mol. Biol. 49, 69–89 (2014).
  5. Giege, R., Sissler, M. & Florentz, C. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 26, 5017–5035 (1998).
  6. Freyhult, E., Moulton, V. & Ardell, D. H. Visualizing bacterial tRNA identity determinants and antideterminants using function logos and inverse function logos. Nucleic Acids Res. 34, 905–16 (2006).
  7. Sowa, S. W. et al. Exploiting post-transcriptional regulation to probe RNA structures in vivo via fluorescence. Nucleic Acids Res. 43, e13 (2015).

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