Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P

The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.     

Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA

Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Preparation of membrane vesicles from isolated myelin. Studies on functional and structural properties

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in ²²Na⁺ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myelin basic protein from 0–150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Effects of pH during recombination of human erythrocyte membrane apoprotein and lipid.

The recombinates from human red cell membrane proteins and lipids resulting from dialysis of the components in 2-chloroethanol against aqueous buffers from pH2-12 have been studied by density gradient centrifugation, polyacrylamide gel electrophoresis and freeze-fracture electron microscopy. Between pH 4 and 10 most of the proteins were found in the recombinates whereas below pH 4 and above pH 10 only part of them were recovered in the lipoprotein band after density gradient centrifugation. At low pH, increasing incorporation of the "major glycoprotein" into the recombinates was detected by gel electrophoresis and in parallel increasing amounts of particles were found in the freeze-fracture membrane faces. The necessity of working at low pH values from pH 2-4, however, and a critical evaluation of all the data presently available leads to the conclusion that the 2-choloroethanol technique is not adequate for recombination studies tending to membrane reconsitution.     

Specialized transduction of the ilvD-thyB-ilvA region mediated by Bacillus subtilis bacteriophage SP beta

Specialized transducing SP beta particles were found that carried the Bacillus subtilis genes lying to the left of the prophage attachment site. Three classes of transducing particles were differentiated, depending upon whether they carried ilvA only, thyB and ilvA, or ilvD, thyB, and ilvA. Lysates prepared by the induction of strains that carried both a transducing phage and a plaque-forming phage contained the two particles in a ratio of about 1:3,000. When the transducing particles were used to transduce a phage-sensitive auxotrophic strain to prototrophy, some of the transductants carried only the transducing phage genomes which, by themselves, were defective. One putative nondefective transducing phage (for ilvA only) is also described. SP beta can mediate specialized transduction even in the absence of the major (recE) bacterial recombination system.     

Insertion of bacteriophage SP beta into the citF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes.

We isolated a strain of Bacillus subtilis in which the SP beta c2 prophage is inserted into the citF (succinate dehydrogenase) gene. Defective specialized transducing particles for the ilvBC-leu genes were isolated from phage-induced lysates of this lysogen. We isolated a group of phages that differ in the amount of genetic material they carry from this region. Also, we incorporated mutant ilv and leu alleles into the genomes of several transducing phages. Our phage collection enables us to identify the cistron of new ilv and leu mutations by complementation analysis. In this process we discovered a fourth leu cistron, leuD. Characterization of the phages confirmed the published gene order: ilvB-ilvC-leuA-leuC-leuB; leuD lies to the right of leuB.     

Phenotypical features of long Q-T syndrome in transgenic mice expressing human Na-K-ATPase alpha(3)-isoform in hearts

To understand why the adult human heart expresses three isoforms of the sodium pump, we generated transgenic mice (TGM) with 2.3- to 5. 5-fold overexpression of the human alpha(3)-isoform of Na-K-ATPase in the heart. Hearts from the TGM had increased maximal Na-K-ATPase activity and ouabain affinity compared with control hearts, even though the density of Na-K-ATPase pump sites (of all isoforms) was similar to that of control mice. In perfused hearts, contractility both at baseline and in the presence of ouabain tended to be greater in TGM than in controls. Surface electrocardiograms in anesthetized TGM had a steeper dependence of Q-T on sinus cycle length, and Q-T intervals measured during atrial pacing were significantly longer in TGM. Q-T dispersion during sinus rhythm also tended to be longer in TGM. Thus TGM overexpressing human alpha(3)-isoform have several of the phenotypical features of human long Q-T syndrome, despite the absence of previously described mutations in Na(+) or K(+) channels.