Search

Splicing of precursor messenger RNA is catalyzed by the spliceosome, a dynamic ribonucleoprotein assembly composed of five small nuclear (sn)RNAs and >100 proteins. RNA components catalyze the two transesterification reactions, but proteins perform critical roles in assembly and rearrangement. The catalytic core comprises a paired complex involving U2 and U6 snRNAs for the major form of the spliceosome and U12 and U6atac snRNAs for the minor variant (~0.3% of all spliceosomes in higher eukaryotes); the latter performs identical chemistry, despite limited sequence conservation outside key catalytic elements, and lack of the multi-stem central junction found in the U2-U6 snRNA complex. Here we use solution NMR techniques to show that base pairing patterns of the U12-U6atac snRNA complex of both human and Arabidopsis share key elements with the major spliceosome’s U2-U6 snRNA complex; probing of the single-stranded segment opposing termini of the snRNAs indicates elongation in this region in place of the stacked base pairs at the base of the U6 intramolecular stem loop in the U2-U6 snRNA complex. Binding affinity of RBM22, a protein implicated in remodeling human U2-U6 snRNA prior to catalysis, to U12-U6atac was analyzed by electrophoretic mobility shift assays in which we monitored migration of both protein and RNA components in the same gel. Results indicate that RBM22 binds the U2-U6 and U12-U6atac snRNA complexes specifically and with Kd = 3.5 µM and 8.2 µM, respectively. Similar affinity between RBM22 and each RNA complex suggests that the protein performs the same role in both spliceosomes.

Intron removal during splicing of precursor pre-mRNA requires assembly of spliceosomal small nuclear (sn)RNAs into catalytically competent conformations to promote two transesterification reactions. U2 and U6 snRNA are the only snRNAs directly implicated in pre-mRNA splicing catalysis, but rearrangement and remodeling steps prior to catalysis require numerous proteins. Previous studies have shown that the protein-free U2-U6 snRNA complex adopts two conformations characterized by four and three helices surrounding a central junction in equilibrium. To analyze the role of the central junction in positioning the two helices critical for formation of the active site, we used ensemble time-resolved fluorescence resonance energy transfer to measure distances between fluorophores at selected locations in constructs representing the protein-free human U2-U6 snRNA complex. Data describing four angles in the four-helix conformer suggest the complex adopts a tetrahedral geometry; addition of Mg2+results in shortening of the distances between neighboring helices, indicating compaction of the complex around the junction. In contrast, the three-helix conformer shows a closer approach between the two helices bearing critical elements, but addition of Mg2+widens the distance between these stems. Presence of Mg2+also enhances the steady state fraction of the three-helix conformer found to be active in spliceosomes. Although the central junction assumes a significant role in orienting helices, in neither conformer, with or without Mg2+, are the critical helices positioned sufficiently close to favor interaction, implying that a major role of spliceosomal remodeling proteins is to overcome such distances to create and stabilize a catalytically active fold.

We describe a method to analyze the affinity and specificity of interactions between proteins and RNA using horizontal PAGE under non-denaturing conditions. The method permits tracking of migration of anionic and cationic biomolecules and complexes toward anode and cathode, respectively, therefore enabling quantification of bound and free biomolecules of different charges and affinity of their intermolecular interactions. The gel is stained with a fluorescent intercalating dye (SYBR®Gold or ethidium bromide) for visualization of nucleic acids followed by Coomassie® Brilliant Blue R-250 for visualizations of proteins; the dissociation constant is determined separately from the intensity of unshifted and shifted bands visualized by each dye. The method permits calculation of bound and unbound anionic nucleic acid and cationic protein components in the same gel, regardless of charge, under identical conditions, and avoids the need for radioisotope or fluorescent labeling of either component.

Optical ruler methods employing multiple fluorescent labels offer great potential for correlating distances among several sites, but are generally limited to interlabel distances under 10 nm and suffer from complications due to spectral overlap. Here we demonstrate a multicolor surface energy transfer (McSET) technique able to triangulate multiple points on a biopolymer, allowing for analysis of global structure in complex biomolecules. McSET couples the competitive energy transfer pathways of Förster Resonance Energy Transfer (FRET) with gold-nanoparticle mediated Surface Energy Transfer (SET) in order to correlate systematically labeled points on the structure at distances greater than 10 nm and with reduced spectral overlap. To demonstrate the McSET method, the structures of a linear B-DNA and a more complex folded RNA ribozyme were analyzed within the McSET mathematical framework. The improved multicolor optical ruler method takes advantage of the broad spectral range and distances achievable when using a gold nanoparticle as the lowest energy acceptor. The ability to report distance information simultaneously across multiple length scales, short-range (10-50 Å), mid-range (50-150 Å), and long-range (150-350 Å), distinguishes this approach from other multicolor energy transfer methods.

Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically excited organic fluorophores to small metal nanoparticles, may be used as a molecular beacon/ruler similar to FRET, but with advantages over this classical technique. Here we use NSET to measure Mg(2+)-induced conformational changes for a hammerhead ribozyme and confirm these measurements using FRET. These optical experiments enhance our understanding of the different kinetic pathways for this ribozyme.

We have investigated electrostatic and surface features of an essential region of the catalytic core of the spliceosome, the eukaryotic precursor messenger (pre-m)RNA splicing apparatus. The nucleophile for the first of two splicing reactions is the 2'-hydroxyl (OH) of the ribose of a specific adenosine within the intron. During assembly of the spliceosome's catalytic core, this adenosine is positioned by pairing with a short region of the U2 small nuclear (sn)RNA to form the pre-mRNA branch site helix. The solution structure of the spliceosomal pre-mRNA branch site [Newby,M.I. and Greenbaum,N.L. (2002) Nature Struct. Biol., 9, 958-965] showed that a phylogenetically conserved pseudouridine (psi) residue in the segment of U2 snRNA that pairs with the intron induces a markedly different structure compared with that of its unmodified counterpart. In order to achieve a more detailed understanding of the factors that contribute to recognition of the spliceosome's branch site helix and activation of the nucleophile for the first step of pre-mRNA splicing, we have calculated surface areas and electrostatic potentials of psi-modified and unmodified branch site duplexes. There was no significant difference between the total accessible area or ratio of total polar:nonpolar groups between modified and unmodified duplexes. However, there was substantially greater exposure of nonpolar area of the adenine base, and less exposure of the 2'-OH, in the psi-modified structure. Electrostatic potentials computed using a hybrid boundary element and finite difference nonlinear Poisson-Boltzmann approach [Boschitsch, A.H. and Fenley, M.O. (2004) J. Comput. Chem., 25, 935-955] revealed a region of exceptionally negative potential in the major groove surrounding the 2'-OH of the branch site adenosine. These surface and electrostatic features may contribute to the overall recognition of the pre-mRNA branch site region by other components of the splicing reaction.

Understanding the process by which RNA molecules fold into stable structures includes study of the role of site-bound metal ions. Because the alkaline earth metal ions typically associated with RNA structure [most often Mg(II)] do not provide convenient spectroscopic signals, replacement with metal ions having spectroscopically useful properties has been a valuable approach. The luminescence properties of the lanthanide(III) series, in particular europium(III), have made them useful in the study of complexation with biomolecules. We review the physical, chemical, and spectroscopic characteristics of Eu(III) that contribute to its value as a probe of RNA-metal ion interactions, and examples of information obtained from studies of Eu(III) bound to small RNA stem loops. Although Eu(III) has similar site preference to Mg(II), luminescence and isothermal titration calorimetry measurements indicate that Ln(III) loses water molecules from the inner hydration sphere more readily than does Mg(II), resulting in more direct coordination between RNA and the metal ion and very different energetics of binding. In some cases, e.g., a GAAA tetraloop, binding appears to occur by a lock and key process; in the same base sequence containing certain deoxynucleoside substitutions that alter loop structure, binding appears to occur by an induced fit process.

Spin labels have been extensively used to study the dynamics of oligonucleotides. Spin labels that are more rigidly attached to a base in an oligonucleotide experience much larger changes in their range of motion than those that are loosely tethered. Thus, their electron paramagnetic resonance spectra show larger changes in response to differences in the mobility of the oligonucleotides to which they are attached. An example of this is 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine (1). How ever, the synthesis of this modified DNA base is quite involved and, here, we report the synthesis of a new spin-labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (2). This spin label is readily prepared in half the number of steps required for 1, and yet behaves in a spectroscopically analogous manner to 1 in oligonucleotides. Finally, it is shown here that both spin labels 1 and 2 can be used to detect the formation of both double-stranded and triplex DNA.

Pairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine residue. The 2' OH of this adenosine, called the branch site, brings about nucleophilic attack at the pre-mRNA 5' splice site in the first step of splicing. Another feature of this pairing is the phylogenetic conservation of a pseudouridine (psi) residue in U2 snRNA nearly opposite the branch site. We show that the presence of this psi in the pre-mRNA branch-site helix of Saccharomyces cerevisiae induces a dramatically altered architectural landscape compared with that of its unmodified counterpart. The psi-induced structure places the nucleophile in an accessible position for the first step of splicing.

Many noncoding RNA molecules adopt alternative secondary and tertiary conformations that are critical for their roles in gene expression. Although many of these rearrangements are mediated by other biomolecular components, it is important to evaluate the equilibrium relationship of the conformers. To measure the spontaneous interconversion in a bi-stable RNA stem loop sequence into which a single 19 F-uridine label was incorporated, a 19 F– 19 F EXSY experiment was employed. The kinetic exchange rate measured from EXSY experiments for this system was 37.3 ± 2.8 s −1 . The advantage of this approach is that exchange kinetics can be monitored in any RNA sequence into which a single 19 F nucleotide is incorporated by commercial synthesis. This method is therefore suitable for application to biologically significant systems in which dynamic conformational rearrangement is important for function and may therefore facilitate studies of RNA structure–function relationships.

Small functional RNAs required for structure studies are often prepared by in vitro transcription. Capillary electrophoresis in liquid crystalline gels of Pluronic F127 was used to analyze unfractionated in vitro transcription reactions and anion-exchange high-performance liquid chromatography (HPLC) fractions from transcription reactions. Guanosine monophosphate (GMP), the four nucleoside triphosphates (NTPs), abortive transcripts, and transcripts with lengths near the desired product length were simultaneously resolved and quantified in a single run. Oligonucleotides up to at least 35 nucleotides were resolved to baseline within 10 min using a moderate field (185 V/cm) and short effective capillary length (7.6 cm) for electrophoresis in 20% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30°C). Nucleotide migration times were 4—5 min, in the order UTP+CTP (unresolved)

Two new NMR structures describing the complex formed by a peptide from the BIV Tat protein with the TAR RNA provide a significant advance in our understanding of the ways in which peptides interact with specific sites in the major groove of their RNA targets.

The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In this study, the first stem loop of the SL1 RNA from the nematode Caenorhabditis elegans was examined by homonuclear and heteronuclear NMR. Results of enzymatic cleavage patterns established that the first 36 nucleotides (which includes the splice site and a complementary base-paired region surrounding a nine-nucleotide hairpin loop) remain structurally independent of the rest of the 100-nucleotide full-length transcript. A comparison of exchangeable and non-exchangeable proton chemical shifts in the region of the splice site and loop between the native sequence and a modified 26-nucleotide fragment from which an asymmetric internal loop had been deleted was made. There was no significant difference between the resonance locations of the equivalent protons in the two molecules, establishing that there was no tertiary interaction between the hairpin and internal loops. Full chemical shift assignments of 1H, 13C, and 15N chemical shifts were obtained for the modified fragment by multidimensional homonuclear and heteronuclear NMR spectroscopy. The stem adopts an A-form helix typical of RNA. The A-type helical conformation of the stem appears to continue for the first three nucleotides of the 5' side of the loop, followed by a guanosine residue in a syn conformation about the glycosidic bond. Base stacking is not seen on the 3' side of the loop. There was no evidence for formation of Watson-Crick base-pairs within the loop, but several long distance NOEs indicated cross-loop contacts, indicative of a structured loop. The final loop residues, an adenine which is conserved among all known nematode SL RNA sequences, adopts an extrahelical conformation.

The branch site helix from Saccharomyces cerevisiae with pseudouridine (ψ) incorporated in a phylogenetically conserved position of U2 snRNA features an extrahelical branch site adenosine (A) that forms a base triple interaction with the minor groove edge of a widely conserved purineU2 strand-pyrimidineintron strand (RU2-Yintron) base pair two positions upstream. In these studies, NMR spectra of a duplex in which 2-aminopurine (2ap), a fluorescent analog of adenine lacking the proposed hydrogen bond donor, was substituted for the branch site A, indicated that the substitution does not alter the extrahelical position of the branch site residue; thus, it appears that a hydrogen bond between the adenine amino group and the R-Y pair is not obligatory for stabilization of the extrahelical conformation. In contrast, reversal of the orientation of AU2-Uintron to UU2-Aintron resulted in an intrahelical position for the branch site A or 2ap. Fluorescence intensity of 2ap substituted for the branch site A with the original RU2-Yintron orientation (AU or GC) was high, consistent with an extrahelical position, whereas fluorescence in helices with the reversed R-Y orientation, or with a mismatched pair (A-U → G•A or U•C), was markedly quenched, implying that the residue was stacked in the helix. The A 5′ to the branch site residue was not extrahelical in any of the duplexes. These findings suggest that the RU2-Yintron base pair orientation in the ψ-dependent branch site helix plays an important role in positioning the branch site A for recognition and/or function.

Porphyrins are transported in the serum bound to proteins and lipoproteins; the particular component(s) to which a porphyrin is bound influences its distribution in the body. In these experiments, the fractions of serum to which several metalloporphyrin inhibitors of the microsomal enzyme heme oxygenase bind were determined. Sn-mesoporphyrin and Sn-protoporphyin were associated almost entirely with serum albumin and Sn-diiododeuteroporphyrin almost completely with the lipoprotein fraction; Zn-mesoporphyrin was associated with all fractions. Serum transport proteins may be targets of photosensitization by photoactive metalloporphyrins. A decrease in the binding constant of bilirubin to Sn-mesoporphyrin-mediated photosensitized human serum albumin (HSA) was observed following illumination at 50 W/m2 in the spectral range of 520-700 nm; there were 2.0 +/- 0.2 bilirubins bound per HSA for samples illuminated120 min; following 180 min illumination, the stoichiometry decreased to 1.5 +/- 0.1 bilirubin per HSA. In a similar experiment with Zn-mesoporphyrin, the porphyrin was fully photooxidized to a nonphotoactive and noninhibiting product after 1 min illumination. The light reaching a porphyrin through human skin would be considerably less than that utilized under these in vitro conditions, and such effects on serum proteins, if demonstrable at all in vivo, would be expected to be far less pronounced than those measured here.

Adrenalectomy causes a decrease in the arginase activity of the liver1,2 and lactating mammary gland2 in the rat. These findings, together with complementary observations on the abilities of various adrenal cortex steroids to restore the depleted tissue arginase levels after adrenalectomy1,2, are of great interest in view of the fact that it is now believed3 that certain cortical hormones promote gluconeo-genesis from protein, a process entailing deamination of amino-acids and the excretion of the nitrogen as urea through the ornithine cycle which, of course, involves the action of arginase.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful biophysical technique that facilitates determination of the three-dimensional structure and interactions of proteins and nucleic acids in solution. A set of NMR experiments identifies spectral signatures corresponding to specific atoms on individual residues in the molecule and determines their order in the primary sequence. Distance and angular relationships are subsequently measured and utilized to supplement standard force-fields used in molecular dynamics (MD)-based protocols to obtain high-resolution structural models. Structure determination is aided by molecular biology and biochemistry to generate samples optimally labelled with NMR-active isotopes; by improvement in the design of NMR instrumentation to allow spectral signatures to be recorded with high sensitivity; by development of efficient techniques to manipulate nuclear spins at the quantum level; and by generation of advanced computer algorithms that allow rapid processing, manipulation and storage of large quantities of NMR data. Key Concepts: Solution NMR is an excellent spectroscopic technique for the determination of the three-dimensional structure of biomolecules at near-physiological conditions. The abundance of chemical and structural information available from NMR spectra derives from the ability to selectively manipulate specific nuclei by customizable sequences of radio-frequency pulses in a large static magnetic field. NMR signals report on the variety of chemical environments experienced by nuclei in biomolecules through unique spectral signatures. The repertoire of information-rich NMR experiments has been expanded by the ability to enrich biomolecules with NMR-active nuclei taking advantage of advances in molecular biology and biochemistry. Nuclear spins in biomolecules form magnetically coupled networks, with characteristic spectral profiles that can be manipulated to extract angular, orientational and distance information for calculation of three-dimensional structural models. Keywords: NMR; proteins; nucleic acids; three-dimensional structure

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.

Metal ions play a crucial role in the conformation and splicing activity of Group II introns. Results from 2-aminopurine fluorescence and solution NMR studies suggest that metal ion binding within the branch site region of native D6 of the Group II intron is specific for alkaline earth metal ions and involves inner sphere coordination. Although Mg 2+ and Ca 2+ still bind to a mutant stem loop sequence from which the internal loop had been deleted, ion binding to the mutant RNA results in decreased, rather than increased, exposure of the branch site residue to solvent. These data further support the role of the internal loop in defining branch site conformation of the Group II intron. The specific bound Mg 2+ may play a bivalent role: facilitates the extrahelical conformation of the branch site and has the potential to act as a Lewis acid during splicing.

Energy transfer from organic fluorophores to small metal nanoparticles is being used as a molecular beacon tool to monitor the kinetic processes of the hammerhead ribozyme. This marks the first time that nanomaterials have been used to monitor ribozyme kinetics. The quantum efficiency of energy transfer from the fluorophore to the gold nanoparticle follows a distance dependence behavior, which allows the real-time characterization of ribozyme complex structure and cleavage kinetics. The rate of cleavage for our ribozyme at pH=6.5 and 37°C is measured to be on the order of 10-2 min-1, which is the correct order of magnitude for similar ribozymes at this pH in the literature.

Carcinogenic aryl hydrazines produce C8-arylated purine adducts. The effect of these adducts on DNA conformation and their role in hydrazine carcinogenesis are unknown. Here, we describe a new synthetic route to produce these adducts that is also compatible with the synthesis of the corresponding phosphoramidites needed for oligonucleotide synthesis. Two oligonucleotides were prepared, an unmodified oligonucleotide, d((5)(')CGCGCGCGCG(3)(')), and a C8-phenylguanine modified oligonucleotide, d((5)(')CGCGCGCGCG(3)(')) (G = 8-phenylguanine). These oligonucleotides were compared using thermal denaturation, circular dichroism, NMR, and molecular modeling. The phenyl modification destabilizes the B DNA form and stabilizes the Z DNA form such that the B:Z ratio is near one under physiological conditions. In light of recent studies that show a role for Z DNA in gene expression and cell transformation, Z DNA stabilization by C8-arylguanine formation from aryl hydrazines may be relevant to their role in carcinogenesis.

The inherent chemical properties of RNA molecules are expanded by posttranscriptional modification of specific nucleotides. Pseudouridine (ψ), the most abundant of the modified bases, features an additional imino group, NH1, as compared with uridine. When ψ forms a Watson–Crick base pair with adenine in an RNA helix, NH1 is positioned within the major groove. The presence of ψ often increases thermal stability of the helix or loop in which it is found [Hall, K. B. & McLaughlin, L. (1992) Nucleic Acids Res. 20, 1883–1889]. X-ray crystal structures of transfer RNAs [e.g., Arnez, J. & Steitz, T. (1994) Biochemistry 33, 7560–7567] have depicted water molecules bridging ψNH1 groups and nearby phosphate oxygen atoms, but direct evidence for this interaction in solution has not been acquired. Toward this end, we have used a rotating-frame Overhauser effect spectroscopy-type NMR pulse sequence with a CLEAN chemical-exchange spectroscopy spin-lock pulse train [Hwang, T.-L., Mori, S., Shaka, A. J. & van Zijl, P. C. M. (1997) J. Am. Chem. Soc. 119, 6203–6204] to test for ψNH1–water cross-relaxation effects within two RNA helices: ( i ) a complementary duplex, in which ψ is not associated with structural change, and ( ii ) an RNA duplex representing the eukaryotic pre-mRNA branch-site helix from Saccharomyces cerevisiae , in which a conserved ψ extrudes the branch-site adenosine from the helix. Our data implicate a water–ψNH1 hydrogen bond both in stabilizing the complementary helix and in favoring formation of the unique structure of the branch-site helix.

The site-specific binding of metal ions maintains an important role in the structure, thermal stability, and function of folded RNA structures. RNA tetraloops of the "GNRA" family (where N = any base and R = any purine), which owe their unusual stability to base stacking and an extensive hydrogen bonding network, have been observed to bind metal ions having different chemical and geometric properties. We have used laser-induced lanthanide luminescence and isothermal titration calorimetry (ITC) to examine the metal-binding properties of an RNA stem loop of the GNRA family. Previous research has shown that a single Eu(III) ion binds the stem loop fragment in a highly dehydrated site with a K(d) of approximately 12 microM. Curve-fitting analysis of the broad luminescence excitation spectrum of Eu(III) upon complexation with the tetraloop fragment indicates the possibility of two microenvironments that do not differ in hydration number. Binding of Eu(III) to the loop was accompanied by positive enthalpic changes, consistent with energetic cost of removal of water molecules and suggesting that the binding is entropically driven. By comparison, binding of Mg(II) or Mn(II) to the RNA loop, or Eu(III) to the DNA analogue of the loop, was associated with exothermic changes, consistent with predominantly outer-sphere coordination. These results suggest specific binding, most probably involving ligands on the 5' side of the loop.

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.

Group II introns are multidomain ribozymes that catalyze their own removal from pre-mRNA. The nucleophile for the first cleavage step is the 2'OH of a specific adenosine within domain 6 (D6), called the branch site. Mechanistic parallels and limited secondary structural similarity with the eukaryotic spliceosome lead many to speculate that the two systems have a common ancestry. We have elucidated structural features of the branch site region and the importance of the internal loop to branch site conformation within D6 of the ai5gamma Group II intron by NMR and fluorescence spectroscopy. Fluorescence experiments in which 2-aminopurine was substituted for the branch site adenosine suggest that the branch site base is exposed to solvent and that this position is enhanced by Mg2+ or Ca2+. Upfield NMR chemical shifts of imino protons of the two uridine residues flanking the branch site adenosine, and an n --> n + 2 NOE between them, suggest a stacked intrahelical conformation of the two uridines. In contrast, results of NMR and 2-aminopurine fluorescence spectra of a mutated D6 from which the internal loop had been deleted suggest a less exposed position of the branch site adenosine, which is likely to form a G-A base pair with the opposing 3'G. These findings describe a model in which the branch site adenosine of D6 is in an extrahelical position, surrounded by two intrahelical bases. The internal loop and divalent metal ions facilitate this motif.

The content of phosphoribosyl pyrophosphate (PPRibP) and of intermediates involved in its synthesis has been measured in the livers of obese (ob/ob) mice 2 months and 3-4 months of age, a period of dynamic growth and marked hepatic hypertrophy and hyperplasia, and comparison made with the values found in the lean age-matched control groups. The total hepatic PPRibP content and PPRibP/mg DNA was significantly increased in the 3- to 4-month-old obese mice relative to both the lean control groups and the 2-month-old obese mice, illustrating the significant changes occurring in the obese mouse liver in this transition period. The change in hepatic PPRibP/mg DNA in the obese mice is positively correlated with age (up to 4 months, body weight 60 g) and with parameters linked to the activity of the pentose phosphate pathway. There is no apparent correlation between PPRibP concentration and inorganic phosphate, the energy status of the cell, or the hepatic PPRibP synthetase (EC 2.7.6.1.) activity. The increase in the bioavailability of PPRibP, which is both a substrate and activator of the de novo and salvage pathways of purine and pyrimidine synthesis, is considered in relation to the increased nucleotide requirement associated with the rise in total hepatic RNA, DNA, and adenine nucleotide in the obese mouse.

Early renal hypertrophy of diabetes is associated with increases in the tissue content of RNA, DNA, and sugar nucleotides involved in the formation of carbohydrate-containing macromolecules. We have previously reported an increase in the activity of enzymes of the de novo and salvage pathways of purine synthesis in early diabetes; the present communication explores the changes in the pathways of pyrimidine synthesis. Measurements have been made of key enzymes of the de novo and salvage pathways at 3, 5, and 14 days after induction of diabetes with streptozotocin (STZ), phosphoribosyl pyrophosphate (PPRibP), and some purine and pyrimidine bases. Carbamoyl-phosphate synthetase II, the rate-limiting enzyme of the de novo route, did not increase in the first 5 days after STZ treatment, the period of most rapid renal growth; a significant rise was seen at 14 days (+38%). Dihydroorotate dehydrogenase, a mitochondrial enzyme, showed the most marked rise (+147%) at 14 days. The conversion of orotate to UMP, catalyzed by the enzymes of complex II, was increased at 3 days (+42%), a rise sustained to 14 days. The salvage route enzyme, uracil phosphoribosyltransferase (UPRTase), showed a pattern of change similar to complex II. The effect of the decreased concentration of PPRibP on the activities of CPSII, for which it is an allosteric activator, and on activities of OPRTase and UPRTase, for which it is an essential substrate, is discussed with respect to the relative Ka and Km values for PPRibP and the possibility of metabolite channeling.

Metalloporphyrin inhibitors of heme oxygenase may also have photosensitizing properties in vivo. To assess photoactivity in serum, the relative ability to mediate photooxidation of tryptophan or other oxidizable targets, presumably by singlet oxygen production, was measured for tin mesopor-phyrin, zinc mesoporphyrin, and zinc deuteroporphyrin bisglycol in aqueous solution and when bound to human serum albumin. While tin mesoporphyrin sensitized at the greatest initial rate in aqueous solution, the zinc compounds sensitized at a greater initial rate in detergent micelles or when bound to albumin. There was minimal alteration of the tin mesoporphyrin during the time course of illumination in the Soret or visible absorption regions. The zinc compounds, however, proved to be extremely photolabile and were extensively destroyed by light; the photooxidized forms were found to be ineffective as inhibitors of heme oxygenase.

The steady-state distribution of inorganic phosphate and malate between the intra- and extramitochondrial spaces was measured in suspensions of nonrespiring and respiring rat liver mitochondria in which the transmembrane pH difference was incrementally varied. In respiration-inhibited mitochondria, the slope of log [Pi]in/[Pi]out (ordinate) versus delta pH approached 2 by either chemical or isotopic determination of [Pi], and the slope of log [malate]in/[malate]out versus delta pH was 2.0 with an extrapolated log [Pi]in/[Pi]out value of 0.3 at delta pH = 0. We conclude that the distribution of Pi and malate for nonrespiring mitochondria were quantitatively consistent with those predicted by exchange of Pi- for OH- (or cotransport with H+) and of malate 2- for Pi2-. In respiring mitochondria using glutamate + malate as substrate, there was very little pH dependence of Pi or malate accumulation (the slopes were less than 0.5) unless n-butylmalonate (inhibitor of Pi-dicarboxylate exchange) was added before the glutamate and malate, in which case the distribution patterns at delta pH less than 0.4 were similar to those in nonrespiring mitochondria. In either case, however, after reaching a maximal value of 1.1, log [Pi]in/[Pi]out did not further increase with increasing delta pH. Thus, in normally metabolizing mitochondria, the distributions of Pi and malate are not directly correlated with the difference in pH across the membrane.

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in frutose 2,6-bisphosphate formation and flux through phosphofructokinase.

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.

1. Cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) and cyclic guanosine 3′, 5′-monophosphate (cyclic GMP) have been estimated in the kidneys of rats. 2. Ten minutes after unilateral nephrectomy there was a threefold increase of cyclic GMP in the remaining kidney, which was accompanied by a moderate fall of cyclic AMP. 3. The changes in cyclic nucleotides in the remaining kidney after unilateral nephrectomy were of short duration. 4. When an anephric rat was cross-circulated with a normal litter-mate, there was an increase of cyclic GMP concentration in the kidneys of the latter, which reached its maximum 10 min after the establishment of the cross circulation. 5. In experiments where one kidney of a litter-mate was transplanted to the neck of another rat, unilateral nephrectomy was not followed by changes of the level of cyclic nucleotides in either the transplanted or the remaining kidney. Bilateral nephrectomy, however, resulted in a marked increase of cyclic GMP in the transplanted kidney. 6. The clamping of the blood vessels to one kidney for periods up to 10 min had the same effect as unilateral nephrectomy on the concentration of cyclic GMP in the remaining kidney. When the clamp was removed and the circulation restored, the concentrations of cyclic nucleotides returned to preoperative levels in both kidneys.

Catecholamines added in vitro in rat brain synaptosomes activate the decarboxylation of glucose radioactively labelled on carbon 1, suggesting an effective activation of the pentose phosphate pathway. Stimulation also occurred with phenazine methosulphate, reduced glutathione and hydrogen peroxide. The activation of the pentose phosphate pathway by 5-hydroxytryptamine, noradrenaline and dopamine is ascribed to the activation of monoamine oxidase, producing both the respective biogenic aldehyde and hydrogen peroxide. Evidence is presented that the further metabolism of the aldehyde by aldehyde reductase and the removal of hydrogen peroxide by glutathione peroxidase both release the limitation of NADP+ availability for the pentose phosphate pathway by leading to the oxidation of NADPH. The relevance of the maintenance of reduced NADP+ on brain is discussed in relation to the metabolism of glutathione and to lipid peroxidation.

Summary The photosynthetic unit is a complex of pigments and proteins coupled to a reaction center where the initial light-driven charge separation of photosynthetic reactions takes place. The ‘size’ of the photosynthetic unit is defined as the number of light-absorbing pigment molecules contributing excitation energy to a reaction center. It is an important parameter in the study of the structure and function of photosynthetic membranes. Several approaches used for relative and absolute measures of photosystem stoichiometry and size are evaluated, and results achieved by these methods are compared. The concept of the absolute size of a photosynthetic unit leads to a simple relation between the size of the oxygen forming unit, the total chlorophyll per O2 (the classical Emerson-Arnold unit), and the quantum requirement for O2 formation. These measurements lead to further understanding of the mechanisms of energy transfer and of the adaptive mechanisms of plants to varied environments.

The effect of developmental growth on the kidney content of phosphoribosyl pyrophosphate PPRibP was studied in rats at ages between the foetal animal and up to 100 days of age. In addition, the effect of short-term diabetes (up to 14 days) on the renal content of PPRibP was studied in immature rats and in adults aged approx. 60 days. The developmental pattern of PPRibP is such that the PPRibP content is lowest in the young rat and increases as the rate of kidney growth slows. In the adult rat, the early kidney hypertrophy of diabetes is accompanied by a fall in PPRibP content and, again, the PPRibP content returns to normal as the rate of kidney hypertrophy diminishes. Induction of diabetes in the immature rat causes a lesser degree of kidney hypertrophy and also a smaller depression of renal PPRibP content. The activity of PPRibP synthetase (EC 2.7.6.1) is not significantly affected by age or diabetes. The changes in PPRibP content are discussed in relation to the generation of ribose 5-phosphate by the pentose phosphate pathway and the utilization of PPRibP for nucleotide synthesis via the ‘de novo’ and salvage pathways.

1. After hypophysectomy, both body and kidney weights fall, but at different rates. The rate at which the kidney decreases in weight is faster than that of the whole body. 2. Seven days after unilateral nephrectomy, the dry weight of the remaining kidney of hypophysectomized rats, with the exception of rats which had been hypophysectomized for 2 days only, was always heavier than the kidney of control hypophysectomized rats of similar body weight. 3. The difference between the dry weight of kidneys of unilaterally nephrectomized hypophysectomized and control hypophysectomized rats increased from 15% in early hypophysectomized (9 days) to about 35% in late hypophysectomized animals (23 days). 4. The implantation of renal cortical cells from 2 day hypophysectomized rats into unilaterally nephrectomized control litter-mates inhibited compensatory renal hypertrophy in the latter. When a similar operation was made using kidney cells from animals which had been hypophysectomized for 23 days, there was no significant inhibition of compensatory renal hypertrophy. 5. The renal contents of adenosine-3′,5′-monophosphate (cyclic AMP) and of guanosine-3′,5′-monophosphate (cyclic GMP) in rats hypophysectomized for 2 days were of the same order as those in normal rats, but were markedly lower in rats hypophysectomized for 23 days. 6. In contrast to what had been observed in normal rats, in hypophysectomized (2 or 23 days) rats, unilateral nephrectomy did not affect significantly the levels of cyclic nucleotides in the remaining kidney. 7. Cross-circulating anephric normal rats with 2 day hypophysectomized animals resulted in an increase of cyclic GMP content in their kidneys. The cross-circulation between anephric normal rats and 23 days hypophysectomized rats had no effect on the level of renal cyclic GMP of the latter. 8. When rats hypophysectomized for either 2 or 23 days and which had been nephrectomized were cross-circulated with normal rats, there were no changes in the content of cyclic GMP in the kidneys of the latter.

The effect of quinolinic acid on the metabolite content of freeze-clamped livers from alloxan-diabetic rats has been studied 1 h after administration of the drug in vivo. Marked increases were found in the hepatic content of malate, citrate, isocitrate, aspartate, pyruvate, and lactate; there were significant decreases in phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in the quinolinate-treated alloxan-diabetic group relative to the untreated diabetic rats. These changes indicated inhibition at phosphoenolpyruvate carboxykinase (EC 4.1.1.32). There was a significant decrease of the content of long-chain acyl-CoA derivatives and of acetyl-CoA and a rise of free CoA in the quinolinic acid-treated group; the calculated cytosolic ATP ADP × P i , value increased following quinolinic acid treatment. The role of the long-chain acyl-CoA derivatives, acting as a component in the sequence of actions leading to the changes reported here, is discussed.

A dose of 25 μM Ca2+ causes half-maximal stimulation of high affinity binding (100 nM) of glutamate to crude synaptic membranes prepared from hippocampus. This facilitation is antagonized by leupeptin (IC50 about 40 μg/ml), an inhibitor of Ca2+-dependent cystein proteinase (E.C. 3.4.22).

1. The distribution of nitrogen in the bodies of rats treated with purified growth hormone for 23 days, and of rats starved for 6 days, has been studied. 2. Some of the extra nitrogen of the growth hormone-treated animals was found deposited in all the tissues examined, with the exception of the heart and the soleus muscle, but the rate at which the individual muscles grew was not uniform. Pectoralis major, masseter, quadriceps, acromiotrapezius, deep spinal muscle, supraspinatus and the diaphragm grew significantly faster than the body as a whole. 3. All the tissues studied, except the masseter and the soleus muscles, were found to lose nitrogen during inanition. It was observed that, with the exception of the masseter muscle, those tissues gaining protein most rapidly under the influence of growth hormone were those that lost it most rapidly during starvation, and conversely. 4. These results reveal the existence of a group of muscles with protein of greater lability than that of the rest of the body tissues. These muscles respond most vigorously to the deposition of protein during treatment with growth hormone, and suffer most depletion of protein during inanition.

The activities of hexokinase, citrate-cleavage enzyme, ;malic enzyme' and NADP-linked isocitrate dehydrogenase have been measured in the adipose tissue, testes and adrenals of normal rats, hypophysectomized rats and hypophysectomized rats treated with either thyroxine or thyroxine plus luteinizing hormone. Hypophysectomy reduced the activity of all four enzymes in all three tissues. Thyroxine alone restored the activity of all four enzymes in adipose tissue towards normal but failed to do so in either testes or adrenals. Thyroxine and luteinizing hormone restored the citrate-cleavage enzyme activity of testes and increased the activity of hexokinase from the low value after hypophysectomy. Neither ;malic enzyme' nor isocitrate dehydrogenase was increased by thyroxine or thyroxine and luteinizing hormone in testes. The differential stimulation of enzyme activity by thyroxine in the different tissues suggests thyroxine as having a special significance in adipose-tissue lipogenesis.

SUMMARY 1. The effect of pH on the diabetogenic action of alloxan has been studied. Dissolving alloxan in buffer of pH 4·0 prior to injection potentiates the diabetogenic action. Conversely, dissolving alloxan in buffer of pH 8·0 decreases the diabetogenic effect. 2. A routine method for the production of diabetes is proposed. The incidence of diabetes was increased from 5 to 93% by the subcutaneous injection of 200 mg alloxan monohydrate/kilo body weight dissolved in citrate phosphate buffer, pH 4·0, instead of in water. 3. Systemic acidosis (either by CaCl2 or NH4Cl ingestion, or the respiration of gas mixtures rich in CO2) increased the susceptibility of rats to alloxan. 4. Measurements have been made of reduced glutathione in blood and liver, and the level of ascorbic acid. No correlation has been found between the changes in the level of these substances and the incidence of diabetes. 5. It is suggested that a relative acidity in the β-cells could be a contributory factor which regulates the susceptibility of these cells to alloxan.

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2′-O-dibutyryl 3′:5′-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [14C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of 14C in various products after 2h of incubation. Fluxes of [14C]acetate, [14C]pyruvate or [14C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolase×triose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD+]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the `malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.