Edward P. Nikonowicz

Solution Conformations of Unmodified and A37N6-dimethylallyl Modified Anticodon Stem-loops of Escherichia coli tRNAPhe

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.

Bacillus anthracis virulence regulator AtxA: oligomeric state, function and CO(2) -signalling.

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 polyacrylamide 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 13 Å. 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. Finally, 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 towards the dimeric state.

Recognition modes of RNA tetraloops and tetraloop-like motifs by RNA-binding proteins

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. WIREs RNA 2014, 5:49–67. doi: 10.1002/wrna.1196

NMR structure and dynamics of the Specifier Loop domain from the Bacillus subtilis tyrS T box leader RNA

Gram-positive bacteria utilize a tRNA-responsive transcription antitermination mechanism, designated the T box system, to regulate expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes. The RNA transcripts of genes controlled by this mechanism contain 5′ untranslated regions, or leader RNAs, that specifically bind cognate tRNA molecules through pairing of nucleotides in the tRNA anticodon loop with nucleotides in the Specifier Loop domain of the leader RNA. We have determined the solution structure of the Specifier Loop domain of the tyrS leader RNA from Bacillus subtilis. Fifty percent of the nucleotides in the Specifier Loop domain adopt a loop E motif. The Specifier Sequence nucleotides, which pair with the tRNA anticodon, stack with their Watson–Crick edges rotated toward the minor groove and exhibit only modest flexibility. We also show that a Specifier Loop domain mutation that impairs the function of the B. subtilis glyQS T box RNA disrupts the tyrS loop E motif. Our results suggest a mechanism for tRNA–Specifier Loop binding in which the phosphate backbone kink created by the loop E motif causes the Specifier Sequence bases to rotate toward the minor groove, which increases accessibility for pairing with bases in the anticodon loop of tRNA.

Solution Nuclear Magnetic Resonance Analyses of the Anticodon Arms of Proteinogenic and Nonproteinogenic tRNA(Gly).

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNA(Gly,GCC) and tRNA(Gly,UCC)), and the third is nonproteinogenic (np-tRNA(Gly,UCC)) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNA(Gly,UCC) with a loop-closing C-A(+) base pair melts at a temperature 10 °C lower than those of tRNA(Gly,GCC) and np-tRNA(Gly,UCC). U-A and C-G pairs close the loops of the latter two molecules and enhance stem stability. Mg(2+) stabilizes the tRNA(Gly,UCC) anticodon arm and reduces the T(m) differential. The structures of the three tRNA(Gly) anticodon arms exhibit small differences among one another, but none of them form the classical U-turn motif. The anticodon loop of tRNA(Gly,GCC) becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNA(Gly,UCC) and np-tRNA(Gly,UCC) establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences.

Conformation Effects of Base Modification on the Anticodon Stem-Loop of Bacillus subtilis tRNA(Tyr).

tRNA molecules contain 93 chemically unique nucleotide base modifications that expand the chemical and biophysical diversity of RNA and contribute to the overall fitness of the cell. Nucleotide modifications of tRNA confer fidelity and efficiency to translation and are important in tRNA-dependent RNA-mediated regulatory processes. The three-dimensional structure of the anticodon is crucial to tRNA-mRNA specificity, and the diverse modifications of nucleotide bases in the anticodon region modulate this specificity. We have determined the solution structures and thermodynamic properties of Bacillus subtilis tRNA(Tyr) anticodon arms containing the natural base modifications N(6)-dimethylallyl adenine (i(6)A(37)) and pseudouridine (ψ(39)). UV melting and differential scanning calorimetry indicate that the modifications stabilize the stem and may enhance base stacking in the loop. The i(6)A(37) modification disrupts the hydrogen bond network of the unmodified anticodon loop including a C(32)-A(38)(+) base pair and an A(37)-U(33) base-base interaction. Although the i(6)A(37) modification increases the dynamic nature of the loop nucleotides, metal ion coordination reestablishes conformational homogeneity. Interestingly, the i(6)A(37) modification and Mg(2+) are sufficient to promote the U-turn fold of the anticodon loop of Escherichia coli tRNA(Phe), but these elements do not result in this signature feature of the anticodon loop in tRNA(Tyr).

Solution NMR determination of hydrogen bonding and base pairing between the glyQS T box riboswitch Specifier domain and the anticodon loop of tRNAGly

In Gram‐positive bacteria the tRNA‐dependent T box riboswitch regulates the expression of many amino acid biosynthetic and aminoacyl‐tRNA synthetase genes through a transcription attenuation mechanism. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson–Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. Enhanced packing within the Specifier domain loop E motif may stabilize the complex and contribute to cognate tRNA selection.

Z-restored spin-echo 13C 1D spectrum of straight baseline free of hump, dip and roll

A pulse sequence of z‐restored spin echo, − π− β− τ− π− τ−, employing a π pulse in the middle of the delay (2τ) to form a spin echo and the two π pulses together to restore the residual longitudinal magnetization back to + z direction, is described. 13C spectra of organic compounds provide a wealth of structural information; however, 13C 1D spectra acquired using reverse geometry probes can have significant baseline humps or rolls because of pulse ring‐down within the coil. The baseline distortions are especially apparent in spectra acquired using cryogenically enhanced probes. The baseline problem may be alleviated by extending the delay between the last pulse and the starting point of acquisition. However, uses of long delay times introduce large negative first‐order phase corrections which themselves produce baseline roll. The prescribed experiment can be used to completely remove the hump, roll or dip in the baseline of the 13C spectrum and at the same time obtain sensitivity similar to the experiment of a single β pulse. We believe that this experiment will be of general applications in acquiring high‐quality 13C NMR data with reverse geometry probes and spectral interpretation. Copyright

Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis

Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.

Comparison of H5 and H8 relaxation rates of a 2H/13C/15N labeled RNA oligonucleotide with selective protonation at C5 and C8

Uniformly 13C/15N enriched ribonucleotide monophosphates have been prepared with extensive deuterium enrichment of the non‐exchangeable positions. The purine C8 and pyrimidine C5 base positions were selectively protonated prior to incorporation of the individual nucleotide triphosphates into an RNA oligonucleotide. The longitudinal and transverse relaxation rates of the H8 and H5 resonances of this deuterated molecule were compared with the relaxation rates of the corresponding protonated, 13C/15N enriched RNA molecule. Deuteration disrupts the efficiency of 1H–1H dipolar relaxation and reduces the longitudinal and transverse magnetization relaxation rates on average to 25% and 68%, respectively, of the values measured for the non‐deuterated RNA molecule. Importantly, the longitudinal relaxation rates remain sufficiently rapid (>1s−1) to permit the use of short recovery delays in multidimensional NMR experiments without significant loss of sensitivity.