This article reports important new understandings in an enzyme/process used by researchers world wide: in vitro transcription by T7 RNA polymerase. While this is the system of choice for RNA synthesis, it is well known to have limitations, a primary one being “nontemplated” 3’ extensions. This is important to the burgeoning field of RNA therapeutics, but plagues researchers throughout basic science and nanotechnology, as well. Reports over two decades have been conflicting and limited in scope. This article brings a new tool, RNA-Seq, to this problem. The RNA-Seq data drive hypotheses that are tested by more conventional tools, but they also indicate a heterogeneity in this process not previously appreciated. This work will prompt development of approaches for mitigating/eliminating this undesired process. While the practical aspects of this work will be interesting to a very wide audience, the results are also of interest to those studying mechanistic elements of transcription.
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.
In this study, the authors have generated new genome-wide tools for functional genomics of Candida albicans. An ORFeome collection has been constructed with 5,099 sequence validated ORFs cloned into a convenient recombinational cloning vector, which represents 83% of the currently annotated coding sequences of C. albicans. This resource is supported by the CandidaOrfDB database, providing information on the individual plasmids and their sequences. The authors also provide a panel of 49 expression vectors that differ in the combination of promoter/selection marker they are carrying and give different options regarding the tagging of the cloned gene product. They demonstrate a novel high-throughput approach to investigate protein-protein interactions in C. albicans. The construction of the ORFeome constitutes a novel and important step towards a systems biology analysis of C. albicans.
HIV/AIDS is a global health threat that requires new insights into viral targets, especially those that resist mutation and promote novel aspects of the viral life cycle. In these respects, the HIV-1 trans-activation response (TAR) RNA is of ongoing interest. In this study, the authors describe the high resolution crystal structure of a laboratory-evolved protein bound to its tightly associated HIV TAR RNA target. The crystal structure of this complex allowed them to determine that only a subset of the ‘evolved’ protein sequences are actually involved in RNA binding, with the preponderance of interactions localized to a short β-hairpin. This observation led them to synthesize a short, stapled peptide that mimics the β-hairpin and attenuates TAR-dependent transcription in HeLa nuclear lysate. This study provides a detailed framework for peptide-mediated TAR recognition that might be generally applicable to target other disease-relevant RNAs.
