NAR’s Breakthrough Articles present high-impact studies answering long-standing questions in the field of nucleic acids research and/or opening up new areas and mechanistic hypotheses for investigation. These articles are chosen by the Editors on the recommendation of Editorial Board Members and Referees. Articles are accompanied by a brief synopsis explaining the findings of the paper and where they fit in the broader context of nucleic acids research. They represent the very best papers published at NAR.
Reconstitution of the CstF complex unveils a regulatory role for CstF-50 in recognition of 3′-end processing signals
This article describe the reconstitution of cleavage stimulation factor (CstF) and physical and structural analysis for its RNA recognition behavior and role in alternative mRNA processing. It demonstrates that hexameric assembly of CstF creates a high affinity platform to target G/U-rich sequences longer than 10nt, also demonstrating CstF-77 is key in boosting the weak activity of its RNA-binding component. The authors describe a potential role for CstF-50 in the recognition of 3’-end processing signals by restriction of dynamic flexibility in the CstF complex rather than direct contacts with the RNA. A crystal structure of a CstF-50/CstF-77 complex provides a molecular basis for the recruitment of CstF-50 by CstF-77. They conclude that the CstF complex serves as the platform to read the two downstream elements (G/U- and U-rich) simultaneously, using CstF-64 as the primary binding module to increase affinity, while CstF-50 confers the ability to engage both signals efficiently.
Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells
The study describes the generation of two suppressor tRNAs that boost the efficiency of the pyrrolysine system for the genetic incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. The tRNAs extend the repertoire of tools applicable to study processes in the live cell. The archaeal (Pyl)/tRNAPyl pair is commonly used for the incorporation of ncAAs into proteins but the system suffers of low ncAA incorporation rate in cells. By screening rationally designed tRNA sets, the authors selected two tRNA variants giving higher amber suppression yields. Compared to the tRNAPyl, the engineered tRNAs feature higher intracellular concentrations and bear distinct posttranscriptional modifications. They demonstrate incorporation of AAs designed for bioorthogonal chemistry into a relevant biological target in live cells. These results open the way to the development of small intramolecular sensors for the study of protein dynamics in the natural cellular environment.
Incorporation of non-natural amino acids into a nascent peptide is a challenging problem. Incorporation of a consecutive sequence of D-amino acids in vitro translation (FIT) systems usually results in low yields and proline has been shown to form peptide bonds slower than other amino acid. Elongation Factor P (EF-P) is a translation factor that stimulates peptide bond formation between proline residues through interactions with the D-arm on tRNAPro. The authors reasoned that if a D-amino acid were charged to a tRNA that is optimized for interactions with EF-Tu and EF-P, then EF-P could enhance incorporation of consecutive D-amino acids. They employed in vitro translation assays with tRNAPro1 and chimeric tRNAs. Maximum D-amino acid incorporation was achieved with EF-P and a tRNA that contained a tRNAPro1 D-arm to facilitate EF-P interaction and a tRNAGluE2 T-stem to enhance EFTu binding. The paper provides a thorough characterization of EF-P stimulated D-amino acid incorporation.