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Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 0.76 µm ± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1. Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.
Lead-optimization strategies for compounds targeting c-Myc G-quadruplex (G4) DNA are being pursued to develop anticancer drugs. Here, we investigate the structure-activity- relationship (SAR) of a newly synthesized series of molecules based on the pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA. Our synthesized series allows modulation of flexible elements with a structurally preserved scaffold. Biological and biophysical analyses illustrate that substituted 5-nitroindole scaffolds bind to the c-Myc promoter G-quadruplex. These compounds downregulate c-Myc expression and induce cell-cycle arrest in the sub-G1/G1 phase in cancer cells. They further increase the concentration of intracellular reactive oxygen species. NMR spectra show that three of the newly synthesized compounds interact with the terminal G-quartets (5′- and 3′-ends) in a 2 : 1 stoichiometry.
Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found “not guilty”. Now, new surprising evidence repeats its request to be the “smoking gun” which reopens the criminal trial revealing the crucial involvement of this tripeptide.
Mitte März des letzten Jahres erreichte die Corona-Pandemie dann auch die Goethe-Universität: Der Lehrbetrieb in den folgenden Semestern wurde und wird hauptsächlich im digitalen Modus durchgeführt, viele Mitarbeiter*innen arbeiten seitdem im Homeoffice, Hygiene- und Abstandsregeln gelten in Räumen und auf Plätzen. In kürzester Zeit mussten Arbeitsabläufe neu organisiert und viele Services auf digitale Prozesse umgestellt werden. Aber das Wichtigste: Die Infektionszahlen konnten gering gehalten werden. Der UniReport hat einige Hochschulangehörige aus Wissenschaft, Verwaltung und Studierendenschaft nach ihren Erfahrungen und Erkenntnissen in diesem außergewöhnlichen Jahr befragt.
We investigated the folding kinetics of G-quadruplex (G4) structures by comparing the K+-induced folding of an RNA G4 derived from the human telomeric repeat-containing RNA (TERRA25) with a sequence homologous DNA G4 (wtTel25) using CD spectroscopy and real-time NMR spectroscopy. While DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favoured off-pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs. DNA G4s, in particular with regard to the conformation around the glycosidic torsion angle χ that uniformly adopts anti conformations for RNA G4s and both, syn and anti conformation for DNA G4s. Modified DNA G4s with 19F bound to C2′ in arabino configuration adopt exclusively anti conformations for χ. These fluoro-modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting the correlation between folding kinetics and pathways with differences in χ angle preferences in DNA and RNA, respectively.
Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.
Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.
2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
G-quadruplexes (G4), found in numerous places within the human genome, are involved in essential processes of cell regulation. Chromosomal DNA G4s are involved for example, in replication and transcription as first steps of gene expression. Hence, they influence a plethora of downstream processes. G4s possess an intricate structure that differs from canonical B-form DNA. Identical DNA G4 sequences can adopt multiple long-lived conformations, a phenomenon known as G4 polymorphism. A detailed understanding of the molecular mechanisms that drive G4 folding is essential to understand their ambivalent regulatory roles. Disentangling the inherent dynamic and polymorphic nature of G4 structures thus is key to unravel their biological functions and make them amenable as molecular targets in novel therapeutic approaches. We here review recent experimental approaches to monitor G4 folding and discuss structural aspects for possible folding pathways. Substantial progress in the understanding of G4 folding within the recent years now allows drawing comprehensive models of the complex folding energy landscape of G4s that we herein evaluate based on computational and experimental evidence.
Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.