Nucleotide Loop Research Articles

Corrinoid salvaging and cobamide remodeling in bacteria and archaea.

Cobamides (Cbas) are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life as co-catalyst of diverse reactions. There are several structural features that distinguish Cbas from one another. The most relevant of those features discussed in this review is the lower ligand, which is the nucleobase of a ribotide located in the lower face of the cyclic tetrapyrrole ring. The above-mentioned ribotide is known as the nucleotide loop, which is attached to the ring by a short linker. In Cbas, the nucleobase of the ribotide can be benzimidazole or derivatives of it, purine or derivatives of it, or phenolic compounds. Given the importance of Cbas in prokaryotic metabolism, it is not surprising that prokaryotes have evolved enzymes that cleave part or the entire nucleotide loop. This function is advantageous when Cbas contain nucleobases that somehow interfere with the function of Cba-dependent enzymes in the organism. After cleavage, Cbas are rebuilt via the nucleotide loop assembly (NLA) pathway, which includes enzymes that activate the nucleobase and the ring intermediate, followed by condensation of activated intermediates and a final dephosphorylation reaction. This exchange of nucleobases is known as Cba remodeling. The NLA pathway is used to salvage Cba precursors from the environment.

NMR structures and magnetic force spectroscopy studies of small molecules binding to models of an RNA CAG repeat expansion.

RNA repeat expansions fold into stable structures and cause microsatellite diseases such as Huntington's disease (HD), myotonic dystrophy type 1 (DM1), and spinocerebellar ataxias (SCAs). The trinucleotide expansion of r(CAG), or r(CAG)exp, causes both HD and SCA3, and the RNA's toxicity has been traced to its translation into polyglutamine (polyQ; HD) as well as aberrant pre-mRNA alternative splicing (SCA3 and HD). Previously, a small molecule, 1, was discovered that binds to r(CAG)exp and rescues aberrant pre-mRNA splicing in patient-derived fibroblasts by freeing proteins bound to the repeats. Here, we report the structures of single r(CAG) repeat motif (5'CAG/3'GAC where the underlined adenosines form a 1×1 nucleotide internal loop) in complex with 1 and two other small molecules via nuclear magnetic resonance (NMR) spectroscopy combined with simulated annealing. Compound 2 was designed based on the structure of 1 bound to the RNA while 3 was selected as a diverse chemical scaffold. The three complexes, although adopting different 3D binding pockets upon ligand binding, are stabilized by a combination of stacking interactions with the internal loop's closing GC base pairs, hydrogen bonds, and van der Waals interactions. Molecular dynamics (MD) simulations performed with NMR-derived restraints show that the RNA is stretched and bent upon ligand binding with significant changes in propeller-twist and opening. Compound 3 has a distinct mode of binding by insertion into the helix, displacing one of the loop nucleotides into the major groove and affording a rod-like shape binding pocket. In contrast, 1 and 2 are groove binders. A series of single molecule magnetic force spectroscopy studies provide a mechanistic explanation for how bioactive compounds might rescue disease-associated cellular phenotypes.

Advanced sampling simulations of coupled folding and binding of phage P22 N-peptide to boxB RNA

Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules’ spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA’s center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes.

From RNA sequence to its three-dimensional structure: geometrical structure, stability and dynamics of selected fragments of SARS-CoV-2 RNA.

In this computational study, we explore the folding of a particular sequence using various computational tools to produce two-dimensional structures, which are then transformed into three-dimensional structures. We then study the geometry, energetics and dynamics of these structures using full electron quantum-chemical and classical molecular dynamics calculations. Our study focuses on the SARS-CoV-2 RNA fragment GGaGGaGGuguugcaGG and its various structures, including a G-quadruplex and five different hairpins. We examine the impact of two types of counterions (K+ and Na+) and flanking nucleotides on their geometrical characteristics, relative stability and dynamic properties. Our results show that the G-quadruplex structure is the most stable among the constructed hairpins. We confirm its topological stability through molecular dynamics simulations. Furthermore, we observe that the nucleotide loop consisting of seven nucleotides is the most flexible part of the RNA fragment. Additionally, we find that RNA networks of intermolecular hydrogen bonds are highly sensitive to the surrounding environment. Our findings reveal the loss of 79 old hydrogen bonds and the formation of 91 new ones in the case when the G-quadruplex containing flanking nucleotides is additionally stabilized by Na+ counterions.

Schizocommunin analogues and derivatives as G-quadruplex ligands and anticancer agents

G-quadruplexes (GQs) have become valid targets for anticancer efforts in the recent couple of decades due to their extremely multifaceted biological functions. Our goal is to quantify interactions within GQs, as well as their interactions with potential ligands (Ls). As secondary nucleic acid (NA) structures, GQs may be understood as a central channel of stacked G-quartets, interlinked into a G-stem (GS) with nucleotide loops. Computational data on full GQ energetics are, however, increasingly noisy. Therefore, we have chosen to simplify our GQ model by stripping it off all nucleotide residues into a “naked” GQ. The GQ-L stacking model allows computing of intrinsic interaction energies, as well as external ligand stacking affinities with “chemical precision”. To relate computed ligand – G4 affinities to their biological activity, we use published inhibitory activities (IC50 values) of several groups of heterocycles. Some of our results provide a good linear relationship between ligand stacking affinities to GQ, calculated by quantum chemical DFT methods, and corresponding log(IC50) values. Herewith we discuss the obtained results in terms of a mechanism of anticancer activity of heterocyclic ligands via complexation with GQs and thereby control of GQ cell regulatory activity.

Understanding the genetic basis of the incompatibility of IncK1 and IncK2 plasmids.

Antimicrobial resistance is a persistent challenge in human and veterinary medicine, which is often encoded on plasmids which are transmissible between bacterial cells. Incompatibility is the inability of two plasmids to be stably maintained in one cell which is caused by the presence of identical or closely related shared determinants between two plasmids originating from partition or replication mechanisms. For I-complex plasmids in Enterobacteriacae, replication- based incompatibility is caused by the small antisense RNA stem-loop structure called RNAI. The I-complex plasmid group IncK consists of two compatible subgroups, IncK1 and IncK2, for which the RNAI differs only by five nucleotides. In this study we focussed on the interaction of the IncK1 and IncK2 RNAI structures by constructing minireplicons containing the replication region of IncK1 or IncK2 plasmids coupled with a kanamycin resistance marker. Using minireplicons excludes involvement of incompatibility mechanisms other than RNAI. Additionally, we performed single nucleotide mutagenesis targeting the five nucleotides that differ between the IncK1 and IncK2 RNAI sequences of these minireplicons. The obtained results show that a single nucleotide change in the RNAI structure is responsible for the compatible phenotype of IncK1 with IncK2 plasmids. Only nucleotides in the RNAI top loop and interior loop have an effect on minireplicon incompatibility with wild type plasmids, while mutations in the stem of the RNAI structure had no significant effect on incompatibility. Understanding the molecular basis of incompatibility is relevant for future in silico predictions of plasmid incompatibility.

Quantifying RNA structures and interactions with a unified reduced chain representation model

RNA is a pivotal molecule that plays critical roles in various cellular processes. Quantifying RNA structures and interactions is essential to understanding RNA function and developing RNA-based therapeutics. Using a unified five-bead model and a non-redundant database, this paper investigates the structural features and interactions of five commonly occurring RNA motifs, i.e., double-stranded helices, hairpin loops, internal/bulge loops, multi-branched junctions, and single-stranded terminal tails. Analyzing detailed distributions of RNA local structural features and base-base interactions reveals a preference for helical structures in both local backbone structures and base orientations. The interactions between adjacent bases exhibit motif-specific and sequence-dependent characteristics, reflecting the distinct topological constraints imposed by different loop-helix connection modes and the varying pairing and stacking interactions among different sequences. These findings shed light on the stability of RNA helices, emphasizing their significance in providing dominant base pairing and stacking interactions for RNA structures and stability. The four non-helix motifs encompass unpaired nucleotide loops and exhibit diverse base-base interactions, contributing to the structural diversity observed in RNA. Overall, the complexity of RNA structure arises from the intricate interplay of base-base interactions.

The coenzyme B12 precursor 5,6-dimethylbenzimidazole is a flavin antagonist in Salmonella.

Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) synthesizes adenosylcobalamin (AdoCbl, CoB12) de novo only under anoxic conditions, but it can assemble the lower ligand loop (a.k.a. the nucleotide loop) and can form the unique C-Co bond present in CoB12 in the presence or absence of molecular oxygen. During studies of nucleotide loop assembly in S. Typhimurium, we noticed that the growth of this bacterium could be arrested by the lower ligand nucleobase, namely 5,6-dimethylbenzimidazole (DMB). Here we report in vitro and in vivo evidence that shows that the structural similarity of DMB to the isoalloxazine moiety of flavin cofactors causes its deleterious effect on cell growth. We studied DMB inhibition of the housekeeping flavin dehydrogenase (Fre) and three flavoenzymes that initiate the catabolism of tricarballylate, succinate or D-alanine in S. Typhimurium. Notably, while growth with tricarballylate was inhibited by 5-methyl-benzimidazole (5-Me-Bza) and DMB, growth with succinate or glycerol was arrested by DMB but not by 5-Me-Bza. Neither unsubstituted benzimidazole nor adenine inhibited growth of S. Typhimurium at DMB inhibitory concentrations. Whole genome sequencing analysis of spontaneous mutant strains that grew in the presence of inhibitory concentrations of DMB identified mutations effecting the cycA (encodes D-Ala/D-Ser transporter) and dctA (encodes dicarboxylate transporter) genes and in the coding sequence of the tricarballylate transporter (TcuC), suggesting that increased uptake of substrates relieved DMB inhibition. We discuss two possible mechanisms of inhibition by DMB.

Synthesis, NMR kinetics and dynamic structure of a 17-mer heptaloop RNA hairpin carrying a 3-N-methyluridine nucleotide residue in the loop region

A 17-mer RNA hairpin (5'GGGAGUXAGCGGCUCCC3') carrying 3-N-methyluridine (m3U) at position X (m3U7-RNA), designed to represent the anticodon stem-loop (ACSL) region of tRNAs to study an open loop state (O-state), was synthesized, purified by HPLC, and characterized by MALDI-ToF_MS and NMR methods. 1H-NMR data revealed primary (P-state in 56.1%), secondary (S-state in 43.9%) and tertiary (∼5-6%) ACSL conformations. Exchange rate constant (kex) for interconversion between P and S states is 112 sec−1 (<Δω ∼454 rad/sec), confirming a slow exchange regime between the two states. Forward (k PS ) and backward (k SP ) rate constants are 49.166 sec−1 and 62.792 sec−1, respectively, leading to a longer life-time (20.339 msec) for the P-state and a shorter life-time (15.926 msec) for the S-state. In accordance with conformational populations determined by 1H-NMR, dynamics of the P/S/tertiary states of m3U7-RNA and its wild-type counterpart (wt-RNA) were studied by three independent MD production simulations. Cluster analysis revealed that wt-RNA reflects the structural characteristics of the ACSL region of tRNAs. The P-state of m3U7-RNA was found to be structurally similar to wt-RNA but lacks an intraloop H-bond between m3U7 and C10 (U33 and nt36 in tRNAs). In the S-state of m3U7-RNA, m3U7 flips out of the loop region. O-state loop conformations of m3U7-RNA were also clustered (4.8%), wherein the loop nucleotides m3U7.A8.G9.C10.G11 stack one after another. We propose that the O-state of m3U7-RNA is the most suitable conformation that makes the loop accessible for complementary nucleotides and for non-enzymatic primordial replication of small circular RNAs. Communicated by Ramaswamy H. Sarma

Understanding the genetic basis of the incompatibility of IncK1 and IncK2 plasmids

Antimicrobial resistance is a persistent challenge in human and veterinary medicine, which is often encoded on plasmids which are transmissible between bacterial cells. Incompatibility is the inability of two plasmids to be stably maintained in one cell which is caused by the presence of identical or closely related shared determinants between two plasmids originating from partition or replication mechanisms. For I-complex plasmids in Enterobacteriacae, replication- based incompatibility is caused by the small antisense RNA stem-loop structure called RNAI. The I-complex plasmid group IncK consists of two compatible subgroups, IncK1 and IncK2, for which the RNAI differs only by five nucleotides. In this study we focussed on the interaction of the IncK1 and IncK2 RNAI structures by constructing minireplicons containing the replication region of IncK1 or IncK2 plasmids coupled with a kanamycin resistance marker. Using minireplicons excludes involvement of incompatibility mechanisms other than RNAI. Additionally, we performed single nucleotide mutagenesis targeting the five nucleotides that differ between the IncK1 and IncK2 RNAI sequences of these minireplicons. The obtained results show that a single nucleotide change in the RNAI structure is responsible for the compatible phenotype of IncK1 with IncK2 plasmids. Only nucleotides in the RNAI top loop and interior loop have an effect on minireplicon incompatibility with wild type plasmids, while mutations in the stem of the RNAI structure had no significant effect on incompatibility. Understanding the molecular basis of incompatibility is relevant for future in silico predictions of plasmid incompatibility.

A potential long-range RNA-RNA interaction in the HIV-1 RNA

It is well-established that viral and cellular mRNAs alike harbour functional long-range intra-molecular RNA-RNA interactions. Despite the biological importance of such interactions, their identification and characterization remain challenging. Here we present a computational method for the identification of certain kinds of long-range intra-molecular RNA-RNA interactions involving the loop nucleotides of a hairpin loop. Using the computational method, we analysed 4272 HIV-1 genomic mRNAs. A potential long-range intra-molecular RNA-RNA interaction within the HIV-1 genomic RNA was identified. The long-range interaction is mediated by a kissing loop structure between two stem-loops of the previously reported SHAPE-based secondary structure of the entire HIV-1 genome. Structural modelling studies were carried out to show that the kissing loop structure not only is sterically feasible, but also contains a conserved RNA structural motif often found in compact RNA pseudoknots. The computational method should be generally applicable to the identification of potential long-range intra-molecular RNA-RNA interactions in any viral or cellular mRNA sequence. Communicated by Ramaswamy H. Sarma

Determining the confromational ensemble of the HIV-1 LTR G-quadruplexes through gaussian-accelerated molecular dynamics using the drude polarizable force field.

Human immunodeficiency virus (HIV) impacts nearly 37.6 million people globally, but there is no effective cure for the infections it causes. HIV-1 is a retrovirus containing a single-stranded RNA genome. After reverse transcription, the 5’ long terminal repeat (LTR) acts as a promoter for viral replication. Two DNA G-quadruplexes (GQs), known as LTR-III and LTR-IV, form within this promoter and inversely regulate viral gene expression. The LTR-III GQ downregulates viral expression and is a quadruplex: duplex hybrid whereas LTR-IV upregulates viral expression and is a parallel GQ containing a single-nucleotide bulge. Their distinct topologies and functional roles suggest their potential to act as selective drug targets for HIV-1.To gain a greater understanding of structure and dynamics of the LTR-III and LTR-IV GQs, we performed conventional molecular dynamics simulations using the Drude polarizable force field. The loop nucleotides in both structures were highly flexible, sampling a variety of conformational states. The LTR-IV loop also formed base pair interactions that limited ion access to its loop cavity and further highlighted two distinct ion binding sites near the backbone tetrad guanines. Within the LTR-III duplex loop, an additional Watson-Crick base pair formed between Ade4:Thy14 and persisted for 40% of the snapshots collected in the total 4-μs simulation time. Analysis of the electric field exerted on Thy14 indicates a preference for base pairing with Ade4 compared to being unpaired as in the NMR ensemble. Gaussian-accelerated molecular dynamics simulations were also performed to identify high and low energy conformational states for subsequent drug targeting. Our results emphasize the importance of electronic polarization in GQ dynamics and can guide future development of antiviral therapeutics.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Sensitive and convenient detection of miRNA-145 using a gold nanoparticle-HCR coupled system: computational and in vitro validations.

Multiple sclerosis (MS) remains a challenging disease that requires timely diagnosis. Therefore, an ultrasensitive optical biosensor based on hybridization chain reaction (HCR) was developed to detect microRNA-145 (miRNA-145) as an MS biomarker. To construct such a sensor, HCR occurred between specific hairpin probes, as MB1 contains a poly-cytosine nucleotide loop and MB2 has a poly-guanine nucleotide sticky end. By introducing miR-145 as a target sequence, long-range dsDNA polymers are formed. Then, positively charged gold nanoparticles (AuNPs) were incubated with the HCR product, which adsorbed onto the dsDNA polymers due to electrostatic adsorption. This resulted in the precipitation of the AuNPs. By incubating different concentrations of miR-145 with AuNPs, the changes in the UV-vis spectrum of the supernatant were analyzed. The proposed biosensor showed a great ability to detect miR-145 in a wide linear range from 1 pM-1 nM with an excellent detection limit (LOD) of 0.519 nM. Furthermore, the developed biosensor indicated considerable selectivity in discriminating between miR-145 and mismatched sequences. It shows high selectivity in differentiating targets. Interestingly, the proposed method was also able to detect miRNA-145 in the diluted serum samples. In conclusion, this sensing platform exhibits high selectivity and specificity for the detection of circulating microRNAs, which holds great promise for translation to routine clinical applications.

Determination of HIV Tropism in Patients with Antiretroviral Therapy Failure in Arkhangelsk Region

The aim of the study was to determine the tropism of the human immunodeficiency virus in patients with virological failure of antiretroviral therapy (ART) from the Arkhangelsk Region based on the analysis of the env gene V3 loop nucleotide sequence.Materials and methods. We used blood plasma samples obtained from 76 HIV-infected persons from the Arkhangelsk Region with virological failure of antiretroviral therapy. The nucleotide sequences of the HIV env gene C2-V3-C3 region were studied by PCR followed by sequencing. The genotype of the studied strains was determined based on the analysis of their phylogenetic relations with reference sequences from the international GenBank database, as well as using specialized programs. To predict viral tropism, the Garrido rule and the online bioinformatic tool Geno2Pheno[coreceptor] were used. The Geno2Pheno[coreceptor] algorithm, determines the false positive rate (FPR) based on the analysis of the env gene V3 loop nucleotide sequence. Results and discussion. Significantly lower representation of R5X4/X4-tropic HIV variants in long-term infected persons with subsubtype A6 virus compared to subtype B virus has been shown. For all FPR cut-off algorithms, a significant correlation between subtype and HIV tropism was observed (p=0.0014 and p=0.013 for FPR 10 % and FPR 20 %, respectively). While among subtype B strains, at least 57 % were identified as R5X4/X4-tropic variants (for an FPR of 10 %), including two strains classified as X4-tropic; among HIV subsubtype A6 even at an FPR of 20 %, the frequency of R5X4/X4-tropic samples only slightly exceeded 22 %. It can be assumed that the dynamics of changes in HIV tropism depends on the virus subtype. Significant differences in the distribution of amino acid residues of the V3 region sequences in the examined group between R5-tropic and R5X4/X4-tropic strains of subsubtype A6 for positions 18 (χ2=7.616, p=0.0058), 21 (χ2=7.281, p=0.007), 24 (χ2=5.587, p=0.0181), and 34 (χ2=5.144, p=0.0233) have been demonstrated. Among the R5X4/X4-tropic strains of the A6 subsubtype, amino acid substitutions were registered at positions 6, 19, 21, 26, 29, 30, which were not found in the R5-tropic A6 strains. The high occurrence frequency of a number of mutations previously described as presumably associated with resistance to maraviroc and similar drugs may indicate a natural polymorphism characteristic of the A6 subsubtype, which does not correlate with resistance to CCR5 co-receptor antagonists.

Structural studies of the phosphoribosyltransferase involved in cobamide biosynthesis in methanogenic archaea and cyanobacteria

Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5′-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5′-phosphate (α-RP) or α-adenylyl-5′-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT.

Acinetobacter baumannii Catabolizes Ethanolamine in the Absence of a Metabolosome and Converts Cobinamide into Adenosylated Cobamides.

ABSTRACTAcinetobacter baumannii is an opportunistic pathogen typically associated with hospital-acquired infections. Our understanding of the metabolism and physiology of A. baumannii is limited. Here, we report that A. baumannii uses ethanolamine (EA) as the sole source of nitrogen and can use this aminoalcohol as a source of carbon and energy if the expression of the eutBC genes encoding ethanolamine ammonia-lyase (EAL) is increased. A strain with an ISAba1 element upstream of the eutBC genes efficiently used EA as a carbon and energy source. The A. baumannii EAL (AbEAL) enzyme supported the growth of a strain of Salmonella lacking the entire eut operon. Remarkably, the growth of the above-mentioned Salmonella strain did not require the metabolosome, the reactivase EutA enzyme, the EutE acetaldehyde dehydrogenase, or the addition of glutathione to the medium. Transmission electron micrographs showed that when Acinetobacter baumannii or Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 synthesized AbEAL, the protein localized to the cell membrane. We also report that the A. baumannii genome encodes all of the enzymes needed for the assembly of the nucleotide loop of cobamides and that it uses these enzymes to synthesize different cobamides from the precursor cobinamide and several nucleobases. In the absence of exogenous nucleobases, the most abundant cobamide produced by A. baumannii was cobalamin.

Evaluation of weak interactions of proteins and organic cations with DNA duplex structures

Molecular interactions and reactions in living cells occur with high background concentrations of organic compounds including proteins. Uncharged water-soluble polymers are commonly used cosolutes in studies on molecular crowding, and most studies argue about the effects of intracellular crowding based on results obtained using polymer cosolutes. Further investigations using protein crowders and organic cations are important in understanding the effects of cellular environments on nucleic acids with negatively charged surfaces. We assessed the effects of using model globular proteins, serum proteins, histone proteins, structurally flexible polypeptides, di- and polyamines, and uncharged polymers. Thermal stability analysis of DNA oligonucleotide structures revealed that unlike conventional polymer cosolutes, basic globular proteins (lysozyme and cytochrome c) at high concentrations stabilized long internal and bulge loop structures but not fully matched duplexes. The selective stabilization of long loop structures suggests preferential binding to unpaired nucleotides in loops through weak electrostatic interactions. Furthermore, the ability of the proteins to stabilize the loop structures was enhanced under macromolecular crowding conditions. Remarkably, the effects of basic proteins on the stability of fully matched duplexes were dissimilar to those of basic amino-acid-rich polypeptides and polyamines. This study provides new insights into the interaction of nucleic acid structures with organic cations.

Investigating the Role of Base‐Triples in the HTLV‐1 pro‐pol Frameshift Site Pseudoknot

The human T‐cell leukemia virus type 1 (HTLV‐1) RNA genome includes two programmed ‐1 ribosomal frameshift sites. Each site includes three RNA elements: a slippery sequence, a spacer, and a structure. We recently determined that the pro‐pol frameshift site contains a pseudoknot structure. Research on other viral frameshift site pseudoknots indicates that contacts between single‐stranded loop nucleotides and base‐pairs in the stems are often critical to pseudoknot function. Although the HTLV‐1 pseudoknot tertiary structure is not determined, base‐triples may form between its two loops and two stems. To test the hypothesis that these tertiary contacts were critical to its function, the frameshift efficiencies were measured for the wild‐type frameshift site and three mutants. Two mutants (A2295U and Loop 2 A to U) were designed to disrupt putative base‐triples forming between loop 2 and stem 1. A third mutant (U2272C) was designed to evaluate the impact of a stem 2 G•U to G‐C substitution. Preliminary data from quadruplicate measurements showed that the frameshift efficiency for all three mutants was moderately increased relative to the wild‐type frameshift site. Whether the changes in the A2295U and Loop 2 A to U mutant frameshift efficiencies were caused by changes to tertiary contacts is hard to determine without complementary methods that establish their presence. Although the increase in the U2272C mutant frameshift efficiency could relate to tertiary contacts, it is more easily explained by a change to the stem 2 thermodynamic stability. Future work will focus on leveraging other methods to evaluate these possibilities.

Maternally transmitted nonsyndromic hearing impairment may be associated with mitochondrial tRNAAla 5601C>T and tRNALeu(CUN) 12311T>C mutations.

BackgroundSequence alternations in mitochondrial genomes, especially in genes encoding mitochondrial tRNA (mt‐tRNA), were the important contributors to nonsyndromic hearing loss (NSHL); however, the molecular mechanisms remained largely undetermined.MethodsA maternally transmitted Chinese pedigree with NSHL underwent clinical, genetic, and biochemical assessment. PCR and direct sequence analyses were performed to detect mitochondrial DNA (mtDNA), GJB2, and SLC26A4 gene mutations from matrilineal relatives of this family. Mitochondrial functions including mitochondrial membrane potential (MMP), ATP, and ROS were evaluated in polymononuclear leukocytes (PMNs) derived from three deaf patients and three controls from this pedigree.ResultsFour of nine matrilineal relatives developed hearing loss at the variable age of onset. Two putative pathogenic mutations, m.5601C>T in tRNAAla and m.12311T>C in tRNALeu(CUN), were identified via PCR‐Sanger sequencing, as well as 34 variants that belonged to mtDNA haplogroup G2b2. Intriguingly, m.5601C>T mutation resided at very conserved nucleotide in the TψC loop of tRNAAla (position 59), while the T‐to‐C substitution at position 12311 located at position 48 in the variable stem of tRNALeu(CUN) and was believed to alter the aminoacylation and the steady‐state level of tRNA. Biochemical analysis revealed the impairment of mitochondrial functions including the significant reductions of ATP and MMP, whereas markedly increased ROS levels were found in PMNs derived from NSHL patients with m.5601C>T and m.12311T>C mutations. However, we did not detect any mutations in GJB2 and SLC26A4 genes.ConclusionOur data indicated that mt‐tRNAAla m.5601C>T and tRNALeu(CUN) 12311T>C mutations were associated with NSHL.