Oligodeoxynucleotide analogs with 5'-linked anthraquinone

We report here the synthesis of novel 5'-linked oligodeoxynucleotides, both normal phosphodiester and phosphorothioate analogs, in which a covalently attached group at the 5'-terminus is an anthraquinone. These compounds represent a new class of antisense compounds in which the base sequence of the oligodeoxynucleotide serves to deliver a nuclease-resistant reactive drug-like molecule to a cellular target nucleic acid (mRNA or DNA).     

Sequences 5'' to translation start regulate expression of petunia rbcS genes.

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes.     

Cell-specific proteins regulate viral RNA translation and virus-induced disease.

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theiler's murine encephalomyelitis virus, requires polypyrimidine tract-binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non-neural cells. The addition of a second-site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue-specific expression and differential RNA-binding properties of PTB and nPTB are important determinants of cell-specific translational control and viral neurovirulence.     

Glutamine and related analogs regulate guanosine monophosphate reductase in Salmonella typhimurium.

The addition of a glutamine analog, 6-diazo-5-oxo-L-norleucine, or an inhibitor of glutamine synthetase, L-methionine-dl-sulfoximine, to the growth media of most Salmonella typhimurium strains resulted in a marked elevation of guanosine monophosphate reductase levels. The elevation caused by either compound required protein synthesis and could be antagonized by exogenous glutamine. In addition, when glutamine auxotrophs were grown in suboptimal concentrations of glutamine, the guanosine monophosphate reductase levels were increased. It is postulated that glutamine or a product of its metabolism may function under normal conditions as a negative regulatory element in the control of guanosine monophosphate reductase and that decreased effective intracellular levels of glutamine result in an increase in the level of the enzyme.     

Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation

Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3–Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.     

RNA switches regulate initiation of translation in bacteria

A large variety of RNA-based mechanisms have been uncovered in all living organisms to regulate gene expression in response to internal and external changes, and to rapidly adapt cell growth in response to these signals. In bacteria, structural elements in the 5' leader regions of mRNAs have direct effects on translation initiation of the downstream coding sequences. The docking and unfolding of these mRNAs on the 30S subunit are critical steps in the initiation process directly modulating and timing translation. Structural elements can also undergo conformational changes in response to environmental cues (i.e., temperature sensors) or upon binding of a variety of trans-acting factors, such as metabolites, non-coding RNAs or regulatory proteins. These RNA switches can temporally regulate translation, leading either to repression or to activation of protein synthesis.     

Structured mRNAs regulate translation initiation by binding to the platform of the ribosome

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.     

Diamino-Antraquinone: A New Intercalating Agent. Synthesis and Linking to Oligodeoxynucleotide

Abstract

Phosphoramidite derivative of 1,4-diamino antraquinone was synthesized, characterized, and incorporated into oligonucleotides. Intercalative interaction between the dye and the nucleic acid was confirmed by CD spectroscopy.     

NOVA1 acts on Impact to regulate hypothalamic function and translation in inhibitory neurons

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New fluorescent cholesterol analogs as membrane probes.

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.