Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs

Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR

Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1′ and C5′ with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg2+ ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly 13C/15N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive 13C–13C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules

Noncanonical G recognition mediates KSRP regulation of let-7 biogenesis

Let-7 is an important tumor-suppressive microRNA (miRNA) that acts as an on-off switch for cellular differentiation and regulates the expression of a set of human oncogenes. Binding of the human KSRP protein to let-7 miRNA precursors positively regulates their processing to mature let-7, thereby contributing to control of cell proliferation, apoptosis and differentiation. Here we analyze the molecular basis for KSRP-let-7 precursor selectivity and show how the third KH domain of the protein recognizes a G-rich sequence in the pre-let-7 terminal loop and dominates the interaction. The structure of the KH3-RNA complex explains the protein recognition of this noncanonical KH target sequence, and we demonstrate that the specificity of this binding is crucial for the functional interaction between the protein and the miRNA precursor

Structure determination of noncanonical RNA motifs guided by 1 H NMR chemical shifts

Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating 1H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6-2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases. © 2014 Nature America, Inc. All rights reserved