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

摘要

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 Mg ~(2+) 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 ~(13) C/ ~(15) N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive ~(13) C- ~(13) C 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.