Exploring Wild-Type and Mutant E. coli Strains for the Synthesis of Site-Specific Labels to Study RNA Structure and Dynamics by NMR

Abstract

Ribonucleic acids are involved in many biological processes including catalysis, transfer and translation of genetic material, and regulation of gene expression. This unique ability to perform a variety of functions, traditionally associated with proteins is largely due to their capacity to adopt three-dimensional structural folds. Studying the 3D architecture of RNAs is critical for not only understanding the molecular basis of RNA function, but will eventually help with structure-assisted drug design, discovery and delivery. Heteronuclear NMR has become a powerful tool for studying the structure and dynamics of RNAs. To date, several RNAs have been well characterized by this method. However, overcrowding of chemical shifts and rapid signal loss in larger RNAs renders current NMR methods ineffectual. To study the structure and dynamics of larger RNAs, the use of site-selectively 13C-labeled nucleotides promises to be very helpful. To synthesize these labels, it was hypothesized that the metabolic pathways of various Escherichia coli wild type and mutant strains are capable of producing specifically labeled nucleotides necessary for making RNA. To test this hypothesis, we evaluated the growth of mutant strains K10-1516 (deficient in glucose-6-phosphate dehydrogenase of the pentose phosphate pathway) and DL323 (deficient in the Krebs cycle) on optimal LeMaster Richards's minimal media supplemented with carbon source 2-13C-glycerol alone or in combination with 13C-formate to enhance labeling at carbon positions bearing protons that are useful for NMR studies. Experimental results showed that the strains produced specific ribose and nucleotide labels that were readily predicted. These labels will enable us to study the structure, function and dynamics of higher size RNA molecules using NMR that would have been otherwise difficult if the commercially available uniformly labeled or unlabeled ribonucleotides were utilized.