R-3 Repository :: Browsing by Author "Nikonowicz, Edward P."

Browsing by Author "Nikonowicz, Edward P."

Now showing 1 - 11 of 11

Capture and Release of tRNA by the T-Loop Receptor in the Function of the T-Box Riboswitch
(American Chemical Society, 2017) Fang, Xianyang; Michnicka, Malgorzata; Zhang, Yikan; Wang, Yun-Xing; Nikonowicz, Edward P.
In Gram-positive bacteria, the tRNA-dependent T-box riboswitch system regulates expression of amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. Binding of uncharged tRNA “closes” the switch, allowing transcription read-through. Structural studies of the 100-nucleotide stem I domain reveal tRNA utilizes base pairing and stacking interactions to bind the stem, but little is known structurally about the 180-nucleotide riboswitch core (stem I, stem III, and antiterminator stem) in complex with tRNA or the mechanism of coupling of the intermolecular binding domains crucial to T-box function. Here we utilize solution structural and biophysical methods to characterize the interplay of the different riboswitch–tRNA contact points using Bacillus subtilis and Oceanobacillus iheyensis glycyl T-box and T-box:tRNA constructs. The data reveal that tRNA:riboswitch core binding at equilibrium involves only Specifier–anticodon and antiterminator–acceptor stem pairing. The elbow:platform stacking interaction observed in studies of the T-box stem I domain is released after pairing between the acceptor stem and the bulge in the antiterminator helix. The results are consistent with the model of T-box riboswitch:tRNA function in which tRNA is captured by stem I of the nascent mRNA followed by stabilization of the antiterminator helix and the paused transcription complex.
Development and structural application of NMR methods for the measurement of residual dipolar couplings and phosphorus-31 chemical shift anisotropy in RNA, using ribosomal helix-35 psi(746) as a model molecule
(2004) O'Neil-Cabello, Erin; Nikonowicz, Edward P.
This work describes how new methods for measuring one-bond, two-bond and three-bond heteronuclear and homonuclear residual dipolar couplings (RDCs) and 31P chemical shift anisotropy (31P CSA) values in RNA molecules of average size have been developed, facilitating the efficient, accurate and precise collection of these previously under-exploited but potentially powerful types of NMR structural constraints. These new methods for measuring RDCs and 31P CSA values have been developed using a 24 nucleotide hairpin derived from helix-35 of the 23S rRNA of E. coli, containing the psi746 modification, as a model molecule. A very large body of these types of NMR constraints has been carefully measured for helix-35psi746 using the novel methods. The residual dipolar coupling constraints can be implemented in structure calculations of RNA molecules, although the resultant structures must be interpreted with caution especially when RDCs from dynamic regions are included. The first round of structure calculations for helix-35psi746 has shown that the RDC constraints measured can have a dramatic impact on improving the quality of this RNA structure.
Investigations into nucleic acid structure and dynamics using heteronuclear nuclear magnetic resonance spectroscopic methods
(2000) DeJong, Eric Scott; Nikonowicz, Edward P.
The high-resolution structures of two separate RNA molecules were determined using heteronuclear nuclear magnetic resonance (NMR) spectroscopy. Both RNA molecules, in their respective systems, perform critical functions of regulating gene expression. To explore the dynamic properties of one molecule, an extensive investigation into its fast (<5ns) intra-molecular motions was carried out. The dynamics investigation was performed using a novel application of heteronuclear relaxation measurements from several base and ribose sites within the molecule. The results of this work were then interpreted using various forms of the Lipari and Szabo motional model. The significance of this research is two fold: (1) it provides detailed structures of two RNA molecules and a basis for their roles in protein recognition and binding and (2) augments the structural characterization with direct measurements of fast internal motions using a novel application of heteronuclear NMR relaxation analysis.
OncomiR-10b hijacks the small molecule inhibitor linifanib in human cancers
(Springer Nature, 2018) Monroig-Bosque, Paloma del C.; Shah, Maitri Y.; Fu, Xiao; Fuentes-Mattei, Enrique; Ling, Hui; Ivan, Cristina; Nouraee, Nazila; Huang, Beibei; Chen, Lu; Pileczki, Valentina; Redis, Roxana S.; Jung, Eun-Jung; Zhang, Xin; Lehrer, Michael; Nagvekar, Rahul; Mafra, Ana Carolina P.; Monroig-Bosque, Maria del Mar; Irimie, Alexandra; Rivera, Carlos; Dumitru, Calin Dan; Berindan-Neagoe, Ioana; Nikonowicz, Edward P.; Zhang, Shuxing; Calin, George A.
The pervasive role of microRNAs (miRNAs) in cancer pathobiology drives the introduction of new drug development approaches such as miRNA inhibition. In order to advance miRNA-therapeutics, meticulous screening strategies addressing specific tumor targets are needed. Small molecule inhibitors represent an attractive goal for these strategies. In this study, we devised a strategy to screen for small molecule inhibitors that specifically inhibit, directly or indirectly, miR-10b (SMIRs) which is overexpressed in metastatic tumors. We found that the multi-tyrosine kinase inhibitor linifanib could significantly inhibit miR-10b and reverse its oncogenic function in breast cancer and liver cancer both in vitro and in vivo. In addition, we showed that the efficacy of linifanib to inhibit tyrosine kinases was reduced by high miR-10b levels. When the level of miR-10b is high, it can "hijack" the linifanib and reduce its kinase inhibitory effects in cancer resulting in reduced anti-tumor efficacy. In conclusion, our study describes an effective strategy to screen for small molecule inhibitors of miRNAs. We further propose that miR-10b expression levels, due to the newly described "hijacking" effect, may be used as a biomarker to select patients for linifanib treatment.
Structural and Functional Studies on the Infectious Salmon Anemia Virus Nucleoprotein
(2013-10-25) Zheng, Wenjie; Tao, Yizhi Jane; Beckingham, Kathleen M.; Nikonowicz, Edward P.; Suh, Junghae; Wang, Qinghua
Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses which are able to cause infectious disease, and even worldwide pandemics. The genome of Orthomyxovirus consists of 6-8 negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes which perform multiple essential functions throughout the virus life cycle. To better understand the structural features of orthomyxovirus RNPs that allow them to be specifically packaged, we performed structural/functional studies of the nucleoprotein (NP), the major protein component of the RNPs, from the infectious salmon anemia virus (ISAV). The crystal structure of the ISAV-NP was determined to 2.7Å resolution. The ISAV-NP possesses a 112-aa N-terminal domain and a bi-lobular core structure that strongly resembles the structure of the influenza virus NP. Because the ISAV-NP forms homogenous dimers that are stable in solution, I was able to study the NP:RNA binding affinity as well as stoichiometry with fluorescence polarization, using recombinant proteins and synthetic oligos. Surprisingly, the RNA binding analysis revealed that each NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. These results suggest that free RNA exists in the orthomyxovirus RNPs, and selective RNP packaging is likely accomplished through direct RNA-RNA interactions.
Structural consequences of base modifications and metal iron interactions on the anticodon stem-loop from tRNA(Phe) (Escherichia coli)
(2002) Cabello-Villegas, Javier; Nikonowicz, Edward P.
The structural effects of naturally modified bases of RNA are mostly uncharacterized. The structural effects of two base modifications and of cations in the anticodon stem loop of tRNAPhe (Escherichia coli) were studied on a 17 mer RNA (ACSLPhe) by NMR spectroscopy. The anticodon region in fully modified tRNAs is proposed to be in a U-turn conformation. Solution NMR studies of unmodified tRNA anticodon stem-loops suggest that base modifications and divalent cations have a minor impact on the structure of the anticodon loop. The unmodified ACSL Phe contains two extra base pairs, 32--38 and 33--37, precluding a U-turn conformation. ACSLPhe contains a tri-nucleotide loop-composed of the anticodon residues. The attachment of a dimethylallyl group at the amino nitrogen of A37 (i6A37) has been proposed to increase the stacking ability of the anticodon residues and residue 37. In the i6A37 containing molecule (i6 A37-ACSLPhe), base pair 33--37 is broken, and base pair 32--38 is destabilized. Several loop resonances enter a regime of intermediate exchange. The presence of Mg2+ has been proposed to stabilize the U-turn in other studies. Mg2+ opens the loop region of ACSLPhe and makes it multi-conformational. Mg 2+ causes a peak pattern that resembles that of i6A37-ACSL Phe and stabilizes the open conformation of i6A37-ACSL Phe. However, i6A37-ACSLPhe in the presence of Mg2+ does not form a stable U-turn. Pseudouridine at position 32 (Psi32) is another naturally occurring modification on tRNAPhe. Psi32 on ACSLPhe (Psi32-ACSL Phe) increases the stability of the stem and is base paired in anti conformation with A38. The structure of Psi32-ACSL Phe is essentially the same as that of ACSLPhe. Two ion probes for Mg2+ binding sites, Mn2+ and Co (NH3)63+, were tested on ACSL Phe and i6A37-ACSLPhe. Both ions bind to the 5' side of the stem regions and weakly to the loop regions. Co (NH3)63+ induces a U-turn conformation in i6A37-ACSLPhe and ACSL Phe but in the latter case a second conformation coexists. It is concluded that the dimethylallyl modification and cations act synergistically in the stabilization of the U-turn. It is proposed that the greater charge density of Co(NH3)63+ relative to Mg2+ is responsible for the formation of a stable U-turn.
Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis
(Oxford University Press, 2014) Davlieva, Milya; Donarski, James; Wang, Jiachen; Shamoo, Yousif; Nikonowicz, Edward P.
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.
Structure and Dynamics of the Tetra-A Loop and (A-A)-U Sequence Motif within the Coliphage GA Replicase RNA Operator
(American Chemical Society, 2017) Chang, Andrew T.; Tran, Michelle; Nikonowicz, Edward P.
The three-dimensional structure of a RNA hairpin containing the RNA operator binding site for bacteriophage GA coat protein is presented. The phage GA operator contains the asymmetric (A-A)-U sequence motif and is capped by a four-adenine (tetra-A) loop. The uridine of the (A-A)-U motif preferentially pairs with the 5′-proximal cross-strand adenine, and the 3′-proximal adenine stacks into the helix. The tetra-A loop is well-ordered with adenine residues 2–4 forming a 3′ stack. This loop conformation stands in contrast to the structure of the 5′-AUUA loop of the related phage MS2 operator in which residues 1 and 2 form a 5′ stack. The context dependence of the (A-A)-U sequence motif conformation was examined using structures of 76 unique occurrences from the Protein Data Bank. The motif almost always has one adenine bulged and the other adenine adopting an A-U base pair. In the case in which the (A-A)-U motif is flanked by only one Watson–Crick base pair, the adenine adjacent to the flanking base pair tends to bulge; 80% of motifs with a 3′ flanking pair have a 3′ bulged adenine, and 84% of motifs with a 5′ flanking pair have a 5′ bulged adenine. The frequencies of 3′- and 5′-proximal adenines bulging are 33 and 67%, respectively, when the (A-A)-U motif is flanked by base pairs on both sides. Although a 3′ flanking cytidine correlates (88%) with bulging of the 5′-proximal adenine, no strict dependence on flanking nucleotide identity was identified for the 5′ side.
The BIophysical Basis for Adaptation: Predicting Evolutionary Outcomes from Physicochemical Properties
(2013-05-13) Benitez Cardenas, Andres; Shamoo, Yousif; Stewart, Charles R.; Nikonowicz, Edward P.; Silberg, Jonathan J.; Segatori, Laura
Experimental evolution can be used in conjunction with biophysical characterization of enzymes to determine the link between cellular fitness and physicochemical properties of enzymes. Sequencing of ancestral and evolved populations can be used to compare the outcomes of experimental evolution with measurements of fitness, using growth rate assays to correlate fitness outcomes to specific mutations. Combined with enzyme assays of kinetic properties that can provide a direct link between genotypic and phenotypic changes of adaptive mutants, we can model the complex relationship between genotypic changes and evolutionary outcomes. Two experimental evolution systems were used to explore the link between enzyme properties and fitness outcomes. In the first series of studies, a “weak link” evolution experiment was used to explore the effect of reducing selection strength on altering accessible pathways for adaptation. In the weak link method the essential gene for adenylate kinase (AK) was replaced in the chromosome of the thermophile Geobacillus stearothermophilus with a homolog from Bacillus subtilis. Replacement with the maladapted gene confers a high fitness cost, and therefore mutations that restore function of AK are strongly favored. Two triple mutants of AK containing a new combination of single point mutants identified under strong selection, AKQ199R/A193V/Q16L and AKQ199R/T179I/Q16L were discovered through an adaptation experiment using a weak temperature ramp; suggesting that the adaptive landscape for AK thermostability is highly constrained. A thermostable coupled assay was developed for measuring adenylate kinase activity using LDHTTHERMOPHILUS and PKGSTEAROTHERMOPHILUS at high temperatures. The triple mutants had increased function compared with the double mutant ancestors, but the triple mutants displayed diminishing returns epistasis on fitness. In the second experimental evolution system, a mathematical model was developed to investigate the role of adaptive mutations, in the tetracycline inactivation enzyme TetX2, on antibiotic resistance to minocycline (MCN). Growth rates measurements, enzyme kinetics, and flux balance equations were used to develop a model to predict the effect on growth rates of TetX2 and seven adaptive TetX2 variants at different MCN concentrations. Population histogram measurements for the experimental evolution study were measured using a high throughput Illumina sequencing method (FREQ-SEQ). We found that the model was able to accurately predict the fitness outcomes for the wild type and the seven single mutants of TetX2 that were originally isolated, as well as for a double mutant that was not used in the development of the original model. The mathematical model accurately predicts that the two mutants TetX2T280A and TetX2N371I provide the largest fitness benefits, in agreement with the results of in vitro experiments on adaptation to MCN. The model was also able to accurately predict enzyme parameters from growth rates values, with a specific emphasis on predicting the ratio of Vmax/KM(MCN). The model allows us to make predictions about the fitness benefits of physicochemical changes to enzymes, and can be used as a high throughput method for determining enzyme kinetic parameters without requiring protein purification. Understanding how physicochemical changes of enzymes relate to phenotypic changes, and ultimately to fitness, requires knowledge of both the molecular basis for determining enzyme properties, and how selection acts on fitness differences to determine evolutionary outcomes. This research provides direct links between physicochemical changes and adaptive phenotypes, as well providing observations of how adaptive landscapes and fitness changes affect evolutionary outcomes.
The Role of Base Modifications on Tyrosyl-tRNA Structure, Stability, and Function in Bacillus subtilis and Bacillus anthracis
(2013-09-16) Denmon, Andria; Nikonowicz, Edward P.; Bennett, George N.; Segatori, Laura; Bartel, Bonnie; Silberg, Joff
tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA).
The T Box Mechanism and Anticodon Stem-Loops: Molecular and Structural Studies of Glycyl-tRNA Anticodon Stem-Loops and Their Binding to the T Box Specifier Domain.
(2013-11-22) Chang, Andrew; Nikonowicz, Edward P.; Bennett, George N.; Landes, Christy F.; Matthews, Kathleen S.; Tao, Yizhi Jane
T box mechanism is a riboswitch commonly used by Gram-positive bacteria to regulate expression of amino-acid related genes such as aminoacyl-tRNA synthetases (aaRS). The T box riboswitch regulates the gene by the mechanism of transcription attenuation. The 5’-UTR of the mRNA forms mutually exclusive anti-terminator or terminator structures depending on whether the tRNA bound is uncharged or charged. This study focuses on the interactions that occur between T box specifier domain (SD) and tRNA anticodon stem-loop (ASL). This intermolecular interaction contributes to the specificity of the T box riboswitch. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. In this study, the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC have been determined. Two of the tRNA molecules are proteinogenic and one is non-proteinogenic and it participates in cell wall biosynthesis. The structures of the three tRNAGly anticodon arms exhibit small differences between one another and there is no evidence that they form the canonical U-turn motif. 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. The NMR data also suggests the ASL may change its structure to form a U-turn when in complex with the T box Specifier domain. Other T box domains also have specific tertiary structure. The NMR data in this study supports the fact that the T box apical loop interacts with the AG loop as seen in the crystal structure. The NMR data suggests that the U70 of the apical loop forms a reversed Hoogsteen A-U base pair with A73 and a U70G mutation has detrimental effects on the structures and interactions between the two loops.

Browsing by Author "Nikonowicz, Edward P."

Results Per Page

Sort Options