A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence. Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media. These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose. The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription. Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC). Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively. These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues. The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs. Examples of several such heteronuclear NMR experiments are shown.

No abstract

Summary AtxA, a unique regulatory protein of unknown molecular function, positively controls expression of the major virulence genes of Bacillus anthracis. The 475-amino acid sequence of AtxA reveals DNA-binding motifs and regions similar to proteins associated with the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). We used strains producing native and functional epitope-tagged AtxA proteins to examine protein-protein interactions in cell lysates and in solutions of purified protein. Co-affinity purification, non-denaturing poly-acrylamide gel electrophoresis, and bis(maleimido)hexane (BMH) cross-linking experiments revealed AtxA homo-multimers. Dimers were the most abundant species. BMH cross-links available cysteines within 13Å. To localize interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions were tested for multimerization and cross-linking. All mutants multimerized, but one mutation, C402S, prevented cross-linking. Thus, BMH uses C402 to make the inter-molecular bond between AtxA proteins, but C402 is not required for protein-protein interaction. C402 is in a region bearing amino acid similarity to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein oligomerization. Lastly, cultures grown with elevated CO2/bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO2/bicarbonate, suggesting that this host-associated signal enhances AtxA function by shifting the dimer/monomer equilibrium toward the dimeric state.

RNA hairpins are the most commonly occurring secondary structural elements in RNAs and serve as nucleation sites for RNA folding, RNA-RNA, and RNA-protein interactions. RNA hairpins are frequently capped by tetraloops, and based on sequence similarity, three broad classes of RNA tetraloops have been defined: GNRA, UNCG, and CUYG. Other classes such as the UYUN tetraloop in histone mRNAs, the UGAA in 16S rRNA, the AUUA tetraloop from the MS2 bacteriophage, and the AGNN tetraloop that binds RNase III have also been characterized. The tetraloop structure is compact and is usually characterized by a paired interaction between the first and fourth nucleotides. The two unpaired nucleotides in the loop are usually involved in base-stacking or base-phosphate hydrogen bonding interactions. Several structures of RNA tetraloops, free and complexed to other RNAs or proteins are now available and these studies have increased our understanding of the diverse mechanisms by which this motif is recognized. RNA tetraloops can mediate RNA-RNA contacts via the tetraloop-receptor motif, kissing hairpin loops, A-minor interactions, and pseudoknots. While these RNA-RNA interactions are fairly well-understood, how RNA binding proteins recognize RNA tetraloops and tetraloop-like motifs remains unclear. In this review, we summarize the structures of RNA tetraloop-protein complexes and the general themes that have emerged on sequence and structure-specific recognition of RNA tetraloops. We highlight how proteins achieve molecular recognition of this nucleic acid motif, the structural adaptations observed in the tetraloop to accommodate the protein binding partner, and the role of dynamics in recognition.

Structured non-coding RNAs underline fundamental cellular processes, but determining their 3D structures remains challenging. We demonstrate herein that integrating NMR 1H chemical shift data with Rosetta de novo modeling can consistently return 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 the experimental structures with high accuracy (0.6 to 2.0 Å all-heavy-atom rmsd) in 18 cases.