-
Biochemistry Feb 2021Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s,...
Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s, Orgel and colleagues reported potentially prebiotic routes to aminoacylated nucleotides and their RNA-templated condensation to form amino acid-bridged dinucleotides. However, it is unclear whether such reactions would have aided or impeded non-enzymatic RNA replication. Determining whether aminoacylated RNAs could have been advantageous in evolution prior to the emergence of protein synthesis remains a key challenge. We therefore tested the ability of aminoacylated RNA to participate in both templated primer extension and ligation reactions. We find that at low magnesium concentrations that favor fatty acid-based protocells, these reactions proceed orders of magnitude more rapidly than when initiated from the -diol of unmodified RNA. We further demonstrate that amino acid-bridged RNAs can act as templates in a subsequent round of copying. Our results suggest that aminoacylation facilitated non-enzymatic RNA replication, thus outlining a potentially primordial functional link between aminoacylation chemistry and RNA replication.
Topics: Aminoacylation; Dinucleoside Phosphates; Nucleic Acid Conformation; Nucleotides; RNA; Templates, Genetic; Transfer RNA Aminoacylation
PubMed: 33523633
DOI: 10.1021/acs.biochem.0c00943 -
Proceedings of the National Academy of... Mar 2020Antibiotic-producing use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and...
Antibiotic-producing use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as and AtaC is monomeric in solution and binds Mn to specifically hydrolyze c-di-AMP to AMP via the intermediate 5'-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in , connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in .
Topics: Adenosine Monophosphate; Bacterial Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Hydrolysis; Mycobacterium tuberculosis; Phosphoric Diester Hydrolases; Second Messenger Systems; Signal Transduction; Streptococcus pneumoniae; Streptomyces
PubMed: 32188788
DOI: 10.1073/pnas.1917080117 -
Nature Communications Oct 2021The nucleotides diadenosine triphosphate (ApA) and diadenosine tetraphosphate (ApA) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase...
The nucleotides diadenosine triphosphate (ApA) and diadenosine tetraphosphate (ApA) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of ApA and ApA and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApAs across the domains of life, but also identify those that are different for ApA or ApA. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.
Topics: Adenosine Triphosphate; Dinucleoside Phosphates; Endoribonucleases; Escherichia coli; Escherichia coli Proteins; HEK293 Cells; Humans; L-Lactate Dehydrogenase; Phosphoglycerate Kinase; Photoaffinity Labels; Protein Binding; Proteomics; Ubiquitin-Activating Enzymes
PubMed: 34608152
DOI: 10.1038/s41467-021-26075-4 -
BMC Evolutionary Biology Feb 2020Human chromosome 19 has many unique characteristics including gene density more than double the genome-wide average and 20 large tandemly clustered gene families. It...
BACKGROUND
Human chromosome 19 has many unique characteristics including gene density more than double the genome-wide average and 20 large tandemly clustered gene families. It also has the highest GC content of any chromosome, especially outside gene clusters. The high GC content and concomitant high content of hypermutable CpG sites raises the possibility chromosome 19 exhibits higher levels of nucleotide diversity both within and between species, and may possess greater variation in DNA methylation that regulates gene expression.
RESULTS
We examined GC and CpG content of chromosome 19 orthologs across representatives of the primate order. In all 12 primate species with suitable genome assemblies, chromosome 19 orthologs have the highest GC content of any chromosome. CpG dinucleotides and CpG islands are also more prevalent in chromosome 19 orthologs than other chromosomes. GC and CpG content are generally higher outside the gene clusters. Intra-species variation based on SNPs in human common dbSNP, rhesus, crab eating macaque, baboon and marmoset datasets is most prevalent on chromosome 19 and its orthologs. Inter-species comparisons based on phyloP conservation show accelerated nucleotide evolution for chromosome 19 promoter flanking and enhancer regions. These same regulatory regions show the highest CpG density of any chromosome suggesting they possess considerable methylome regulatory potential.
CONCLUSIONS
The pattern of high GC and CpG content in chromosome 19 orthologs, particularly outside gene clusters, is present from human to mouse lemur representing 74 million years of primate evolution. Much CpG variation exists both within and between primate species with a portion of this variation occurring in regulatory regions.
Topics: Animals; Base Composition; Base Sequence; Chromosomes; Chromosomes, Human, Pair 19; Conserved Sequence; CpG Islands; DNA Methylation; Dinucleoside Phosphates; Genome; Humans; Lemur; Mice; Multigene Family; Phylogeny; Primates; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid
PubMed: 32106815
DOI: 10.1186/s12862-020-1595-9 -
Biochimica Et Biophysica Acta. General... Nov 2021Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional...
Biochemical, crystallographic and biophysical characterization of histidine triad nucleotide-binding protein 2 with different ligands including a non-hydrolyzable analog of Ap4A.
BACKGROUND
Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet.
METHODS
An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2'-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution.
RESULTS
HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2'-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar K and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors.
CONCLUSIONS
Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2.
SIGNIFICANCE
Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.
Topics: Dinucleoside Phosphates; Humans; Ligands; Mitochondrial Proteins
PubMed: 34329705
DOI: 10.1016/j.bbagen.2021.129968 -
Science Signaling Sep 2022To colonize the host and cause disease, the human enteropathogen must sense, respond, and adapt to the harsh environment of the gastrointestinal tract. We showed that...
To colonize the host and cause disease, the human enteropathogen must sense, respond, and adapt to the harsh environment of the gastrointestinal tract. We showed that the production and degradation of cyclic diadenosine monophosphate (c-di-AMP) were necessary during different phases of growth, environmental adaptation, and infection. The production of this nucleotide second messenger was essential for growth because it controlled the uptake of potassium and also contributed to biofilm formation and cell wall homeostasis, whereas its degradation was required for osmotolerance and resistance to detergents and bile salts. The c-di-AMP binding transcription factor BusR repressed the expression of genes encoding the compatible solute transporter BusAA-AB. Compared with the parental strain, a mutant lacking BusR was more resistant to hyperosmotic and bile salt stresses, whereas a mutant lacking BusAA was more susceptible. A short exposure of cells to bile salts decreased intracellular c-di-AMP concentrations, suggesting that changes in membrane properties induce alterations in the intracellular c-di-AMP concentration. A strain that could not degrade c-di-AMP failed to persist in a mouse gut colonization model as long as the wild-type strain did. Thus, the production and degradation of c-di-AMP in have pleiotropic effects, including the control of osmolyte uptake to confer osmotolerance and bile salt resistance, and its degradation is important for host colonization.
Topics: Animals; Bacterial Proteins; Bile Acids and Salts; Clostridioides; Clostridioides difficile; Dinucleoside Phosphates; Humans; Mice
PubMed: 36067333
DOI: 10.1126/scisignal.abn8171 -
The Journal of Physical Chemistry. B Aug 2020The fluorescence of dinucleotide NADH has been exploited for decades to determine the redox state of cells and tissues and . Particularly, nanosecond (ns) fluorescence...
The fluorescence of dinucleotide NADH has been exploited for decades to determine the redox state of cells and tissues and . Particularly, nanosecond (ns) fluorescence lifetime imaging microscopy (FLIM) of NADH (in free vs bound forms) has recently offered a label-free readout of mitochondrial function and allowed the different "pools" of NADH to be distinguished in living cells. In this study, the ultrafast fluorescence dynamics of NADH-dehydrogenase (MDH/LDH) complexes have been investigated by using both a femtosecond (fs) upconversion spectrophotofluorometer and a picosecond (ps) time-correlated single photon counting (TCSPC) apparatus. With these enhanced time-resolved tools, a few-picosecond decay process with a signatory spectrum was indeed found for bound NADH, and it can best be ascribed to the solvent relaxation originating in "bulk water". However, it is quite unlike our previously discovered ultrafast "dark" component (∼26 ps) that is prominent in free NADH ( , , 18-21). For these two critical protein-bound NADH exemplars, the decay transients lack the ultrafast quenching that creates the "dark" subpopulation of free NADH. Therefore, we infer that the apparent ratio of free to bound NADH recovered by ordinary (>50 ps) FLIM methods may be low, since the "dark" molecule subpopulation (lifetime too short for conventional FLIM), which effectively hides about a quarter of free molecules, is not present in the dehydrogenase-bound state.
Topics: Binding Sites; Dinucleoside Phosphates; Microscopy, Fluorescence; NAD; Oxidoreductases
PubMed: 32660250
DOI: 10.1021/acs.jpcb.0c04835 -
The c-di-AMP-binding protein CbpB modulates the level of ppGpp alarmone in Streptococcus agalactiae.The FEBS Journal Jun 2023Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly...
Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.
Topics: Carrier Proteins; Streptococcus agalactiae; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Bacterial Proteins; Cyclic AMP; Dinucleoside Phosphates; Potassium
PubMed: 36629470
DOI: 10.1111/febs.16724 -
Journal of Virology Sep 2021Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate...
Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging . Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a nearinfrared fluorescent protein (iRFP) upstream of . HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression correlated initially with plasma viremia, expression decreased after 4 to 5 weeks despite high plasma viremia. The reporter genes were codon optimized to remove cytosine/guanine (CG) dinucleotides, and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication and reporter expression and . Both codon-optimized reporter viruses could be visualized during coinfection and reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole-body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.
Topics: Animals; CD4-Positive T-Lymphocytes; Dinucleoside Phosphates; Gene Expression; Genes, Reporter; HIV Infections; HIV-1; Humans; Luciferases; Luminescent Measurements; Luminescent Proteins; Mice; Optical Imaging; Viremia; Virus Replication; Whole Body Imaging
PubMed: 34232063
DOI: 10.1128/JVI.00449-21 -
Stresses that Raise NpA Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacterial RNA.Molecular Cell Sep 2019Present in all realms of life, dinucleoside tetraphosphates (NpNs) are generally considered signaling molecules. However, only a single pathway for NpN signaling has...
Present in all realms of life, dinucleoside tetraphosphates (NpNs) are generally considered signaling molecules. However, only a single pathway for NpN signaling has been delineated in eukaryotes, and no receptor that mediates the influence of NpNs has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular NpN concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5' caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli. ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated NpN concentrations in bacteria may result from widespread Np capping, leading to altered RNA stability and consequent changes in gene expression.
Topics: Acid Anhydride Hydrolases; Dinucleoside Phosphates; Escherichia coli K12; Escherichia coli Proteins; RNA Stability; RNA, Bacterial
PubMed: 31178354
DOI: 10.1016/j.molcel.2019.05.031