Positions 13 and 914 in Escherichia coli 16S ribosomal RNA are involved in the control of translational accuracy.

Using a conditional expression system with the temperature-inducible lambda PL promoter, we previously showed that the single mutations 13U-->A and 914A-->U, and the double mutation 13U-->A and 914A-->U in Escherichia coli 16S ribosomal RNA impair the binding of streptomycin (Pinard et al., The FASEB Journal, 1993, 7, 173-176). In this study, we found that the two single mutations and the double mutation increase translational fidelity, reducing in vivo readthrough of nonsense codons and frameshifting, and decreasing in vitro misincorporation in a poly(U)-directed system. Using oligodeoxyribonucleotide probes which hybridize to the 530 loop and to the 1400 region of 16S rRNA, two regions involved in the control of tRNA binding to the A site, we observed that the mutations in rRNA increase the binding of the probe to the 530 loop but not to the 1400 region. We suggest that the mutations at positions 13 and 914 of 16S rRNA induce a conformational rearrangement in the 530 loop, which contributes to the increased accuracy of the ribosome.     

The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.     

Alignment of the two domains of the hairpin ribozyme-substrate complex defined by interdomain photoaffinity crosslinking

The hairpin ribozyme-substrate complex contains two independently folding domains that interact with one another to form a catalytic complex. However, little is known about the key structural elements involved in these tertiary interactions. Here, we report the use of a photochemical crosslinking method to investigate the relative proximity and orientation of the two domains of the hairpin ribozyme. This method allows the incorporation of a photochemical azidophenacyl group at specified positions within synthetic oligoribonucleotides. Photocrosslinking was performed following the assembly of four RNA oligonucleotides into active ribozyme-substrate complexes. Two photoagent attachment sites in the substrate binding strand within domain A (between positions A7-G8 and A10-G11) and three in the 5' strand of domain B (A20-G21, A22-A23 and A24-C25) were studied. Several crosslinks between the substrate binding strand and the 5' segment of domain B were detected. All of the photo agent-specific crosslinked species were dependent upon proper assembly and folding of the ribozyme-substrate complex. In addition, a substrate base mutation (G+1 to A+1) that prevents the docking of the two domains, blocks the crosslink formation. Four interdomain crosslinks (A7-G8/C25-A26 (two species); A10-G11/A22 and A24-C25/C12-G13) have been shown to retain catalytic activity. Taken together, these results indicate that the characterized crosslinks provide important information concerning the alignment of the two domains and accurately reflect the active docked conformation of the molecule.     

Structural basis for the guanosine requirement of the hairpin ribozyme

To form a catalytically active complex, the essential nucleotides of the hairpin ribozyme, embedded within the internal loops of the two domains, must interact with one another. Little is known about the nature of these essential interdomain interactions. In the work presented here, we have used recent topographical constraints and other biochemical data in conjunction with molecular modeling (constraint-satisfaction program MC-SYM) to generate testable models of interdomain interactions. Visual analysis of the generated models has revealed a potential interdomain base pair between the conserved guanosine immediately downstream of the reactive phosphodiester (G(+1)) and C(25) within the large domain. We have tested this former model through activity assays, using all 16 combinations of bases at positions +1 and 25. When the standard ribozyme was used, catalytic activity was severely suppressed with substrates containing U(+1), C(+1), or A(+1). Similarly, mutations of the putative pairing partner (C(25) to A(25) or G(25)) reduce activity by several orders of magnitude. The U(25) substitution retains a significant level of activity, consistent with the possible formation of a G.U wobble pair. Strikingly, when combinations of Watson-Crick (or wobble) base pairs were introduced in these positions, catalytic activity was restored, strongly suggesting the existence of the proposed interaction. These results provide a structural basis for the guanosine requirement of this ribozyme and indicate that the hairpin ribozyme can now be engineered to cleave a wider range of RNA sequences.     

4-(3,4-dihydro-1H-isoquinolin-2yl)-pyridines and 4-(3,4-dihydro-1H-isoquinolin-2-yl)-quinolines as potent NR1/2B subtype selective NMDA receptor antagonists

A series of 4-(3,4-dihydro-1H-isoquinolin-2yl)-pyridines and analogous quinolines was prepared and evaluated as NR1/2B subtype selective NMDA receptor antagonists. 2-Hydroxyalkylamino substitution combines high affinity with selectivity (vs alpha1 and M1 receptors) and activity in vivo.     

The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.     

A novel regulatory element in the dnmt1 gene that responds to co-activation by Rb and c-Jun

Rb, c-Jun and dnmt1 play critical roles in the process of cellular differentiation. We demonstrate that a regulatory region of murine dnmt1 contains an element which is responsible for transactivation by Rb and c-Jun in P19 embryocarcinoma cells which is not observed in Y1 adrenocarcinoma cells. During differentiation of P19 cells, the induction of Rb and c-Jun coincides with an increase of dnmt1 mRNA. Using linker scanning mutagenesis we identify the element that is responsible for this activation to be a non-canonical AP-1 site. Our data is an example of how a proto-oncogene activates its downstream effectors by recruiting a tumor suppressor. This interaction of Rb and a proto-oncogene might play an important role in differentiation. The responsiveness of dnmt1 to this type of signal is consistent with an important role for regulated expression of dnmt1 during cellular differentiation.     

Normal conjunctival flora in the North American opossum (Didelphis virginiana) and raccoon (Procyon lotor)

We documented the normal conjunctival bacterial flora from 17 opossums (Didelphis virginiana) and 10 raccoons (Procyon lotor) trapped in Manhattan, Kansas (USA) from November 1999 to January 2000. Both raccoons and opossums were free of apparent ocular disease. The inferior conjunctival sacs of each animal were swabbed for aerobic bacterial and Mycoplasma culture and polymerase chain reaction (PCR) for Mycoplasma and Chlamydia detection. All conjunctival samples were positive for one or more species of aerobic bacteria. The most common isolate from opossums was Staphylococcus spp. Other isolates included Streptococcus spp., Bacillus spp., Corynebacterium spp., and Enterococcus faecalis. The most common isolates in raccoons was Bacillus spp. Other isolates included Streptococcus spp., Staphylococcus spp., non-hemolytic Escherichia coli, and Enterococcus faecalis. Mycoplasma culture was negative in samples from opossums and raccoons. Evidence of Mycoplasma and Chlamydia presence was detected by PCR.     

2-(3,4-Dihydro-1H-isoquinolin-2yl)-pyridines as a novel class of NR1/2B subtype selective NMDA receptor antagonists

Recently, we disclosed 4-aminoquinolines as structurally novel NR1/2B subtype selective NMDA receptor antagonists. We would now like to report our findings on structurally related pyridine analogues. The SAR developed in this series resulted in the discovery of high affinity antagonists which are selective (vs alpha1 and M1 receptors) and active in vivo.