Waiting room oral rehydration in the paediatric emergency department.

Oral rehydration is well established in the treatment of acute gastroenteritis, however it is profoundly underutilised as a treatment in the hospital setting. We introduced a protocol of waiting room oral rehydration for children presenting to the Paediatric Emergency Department with vomiting and/or diarrhoea. These children were given oral rehydration from the time of triage prior to medical assessment. During the study period, 251 children presented 269 times with vomiting and/or diarrhoea, of which 205 (76%) were diagnosed with acute gastroenteritis. A similar period 1 year previously was used as comparison, during which 129 children were diagnosed with acute gastroenteritis. During the study period, 58 children (28%) were given intravenous fluids and 47 (23%) were admitted, compared with 72 (56%) given intravenous fluids and 42 (32%) admitted in the comparison group. This protocol is now part of our routine management of children presenting with symptoms of acute gastroenteritis. Waiting room oral rehydration is a simple yet successful intervention that can be implemented in any Emergency Department.

Evaluation of two gene-silencing constructs for resistance to tomato yellow leaf curl viruses in Nicotiana benthamiana plants.

Infiltration of Agrobacterium tumefaciens into intact plant leaves of N. benthamiana was used to test the efficiency of two virus-based silencing constructs conferring resistance to the closely related begomoviruses. The constructs contained the most conserved sequences of the coat protein (CP) gene and replication-associated protein (Rep) gene of Tomato yellow leaf curl Sardinia virus (Sicily strain) (TYLCSV-[Sic]). Both constructs formed a hairpin structure that enhanced the post-transcriptional gene-silencing mechanism. When agro-infiltrated plants were challenged separately with infectious viruses TYLCSV-[Sic] and Tomato yellow leaf curl virus (TYLCV), the plants showed resistance to TYLCSV-[Sic], but not to the related TYLCV.

Inhibition of DNA transcription using cationic mixed monolayer protected gold clusters.

Efficient recognition of DNA is a prerequisite for the development of biological effectors, including transcription and translation regulators, transfection vectors, and DNA sensors. To provide an effective scaffold for multivalent interactions with DNA, we have fabricated mixed monolayer protected gold clusters (MMPCs) functionalized with tetraalkylammonium ligands that can interact with the DNA backbone via charge complementarity. Binding studies indicate that the MMPCs and DNA form a charge-neutralized, nonaggregated assembly. The interactions controlling these assemblies are highly efficient, completely inhibiting transcription by T7 RNA polymerase in vitro.

Structure in nascent RNA leads to termination of slippage transcription by T7 RNA polymerase.

T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA., T7 RNA polymerase will synthesize a 'ladder' of poly-G RNA products. At each step, the ratio of elongation to product release is consistently approximately 0.75 until the RNA reaches a length of approximately 13-14 nt, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore becomes sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7-deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination.

Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase.

The various kinetic and thermodynamic models for transcription elongation all require an understanding of the nature of the melted bubble which moves with the RNA polymerase active site. Is the general nature of the bubble system-dependent or are there common energetic requirements which constrain a bubble in any RNA polymerases? T7 RNA polymerase is one of the simplest RNA polymerases and is the system for which we have the highest-resolution structural information. However, there is no high-resolution information available for a stable elongation complex. In order to directly map melted regions of the DNA in a functionally paused elongation complex, we have introduced fluorescent probes site-specifically into the DNA. Like 2-aminopurine, which substitutes for adenine bases, the fluorescence intensity of the new probe, pyrrolo-dC, which substitutes for cytosine bases, is sensitive to its environment. Specifically, the fluorescence is quenched in duplex DNA relative to its fluorescence in single-stranded DNA, such that the probe provides direct information on local melting of the DNA. Placement of this new probe at specific positions in the non-template strand shows clearly that the elongation bubble extends about eight bases upstream of the pause site, while 2-aminopurine probes show that the elongation bubble extends only about one nucleotide downstream of the last base incorporated. The positioning of the active site very close to the downstream edge of the bubble is consistent with previous studies and with similar studies of the promoter-bound, pre-initiation complex. The results show clearly that the RNA:DNA hybrid can be no more than eight nucleotides in length, and characterization of different paused species suggests preliminarily that these dimensions are not sequence or position dependent. Finally, the results confirm that the ternary complex is not stable with short lengths of transcript, but persists for a substantial time when paused in the middle or at the (runoff) end of duplex DNA.

Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: a new kinetic model.

In order to begin to understand the mechanism of the initiation of transcription in the model bacteriophage T7 RNA polymerase system, the simplest possible reaction, the synthesis of a dinucleotide, has been followed by quench-flow kinetics and numerical integration of mechanism-specific rate equations has been used to test specific kinetic models. In order to fit the observed time dependence in the pre-steady-state kinetics, a model for dinucleotide synthesis is proposed in which rebinding of the dinucleotide to the enzyme-DNA complex must be included. Separate reactions using dinucleotide as a substrate confirm this mechanism and the determined rate constants. The dinucleotide rebinding observed as inhibition under these conditions forms a productive intermediate in the synthesis of longer transcripts, and must be included in future kinetic mechanisms. The rate-limiting step leading to product formation shows a substrate dependence consistent with the binding of two substrate GTP molecules, and at saturating levels of GTP, is comparable in magnitude to the product release rate. The rate of product release shows a positive correlation with the concentration of GTP, suggesting that the reaction shows base-specific substrate activation. The binding of another substrate molecule, presumably via interaction with the triphosphate binding site, likely facilitates displacement of the dinucleotide product from the complex.

Evidence for DNA bending at the T7 RNA polymerase promoter.

Phage T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high-resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. Here, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A complementary set of experiments employing differential phasing from an array of phased A-tracts provides further evidence for both intrinsic and polymerase-induced bends in the T7 RNA polymerase promoter DNA. The bend in the complex is predicted to be about 40-60 degrees and to be centered around positions -2 to +1, at the start site for transcription, while the intrinsic bend is much smaller (about 10 degrees ). These results, viewed in the light of a recent crystal structure for the complex, suggest a mechanism by which binding leads directly to bending. Bending at the start site would then facilitate the melting necessary to initiate transcription.

Positioning of the start site in the initiation of transcription by bacteriophage T7 RNA polymerase.

The determination of various polymerase structures has sparked interest in understanding how the polynucleotide template interacts with the active site. In the primer-independent initiation of transcription, an additional question arises as to how the complex directs the first two bases of the template uniquely into the active site. Recent studies in the model RNA polymerase from bacteriophage T7 demonstrate that upstream duplex contacts provide at least some of the binding specificity and suggest that the enzyme interacts with the template strand in a melted context near the start site for transcription. The current work probes the role of the template strand in positioning of the first two templating bases during initiation. The results suggest that such positioning is not rate-limiting in steady-state turnover, and that the insertion of a very large and flexible linker three or four bases upstream of the start site has no significant effect on the fidelity of start site selection. The insertion of linkers immediately adjacent to the start site, however, does significantly decrease the fidelity of start site selection (as evidenced by a large increase in misinitiation at position +2, with little change in the observed rate of correct initiation), suggesting that some of the non-transcribed template DNA does help to position the first two templating bases into the active site of the RNA polymerase. Finally, incorporation of an abasic site at position -1 yields a similar decrease in initiation fidelity, suggesting a role for stacking of the bases at positions -1 and +1.

Identification of a minimal binding element within the T7 RNA polymerase promoter.

The T7 RNA polymerase promoter has been proposed to contain two domains: the binding region upstream of position -5 is recognized through apparently traditional duplex contacts, while the catalytic domain downstream of position -5 is bound in a melted configuration. This model is tested by following polymerase binding to a series of synthetic oligonucleotides representing truncations of the consensus promoter sequence. The increase in the fluorescence anisotropy of a rhodamine dye linked to the upstream end of the promoter provides a very sensitive measure of enzyme binding in simple thermodynamic titrations, and allows the determination of both increases and decreases in the dissociation constant. The best fit value of Kd=4.0 nM for the native promoter is in good agreement with previous fluorescence and steady state measurements. Deletion of the downstream DNA up to position -1 or to position -5 leads to a fivefold increase in binding, while further sequential single-base deletions upstream result in 20 and 500-fold decreases in binding. These results indicate that the (duplex) region of the promoter upstream of and including position -5 is both necessary and sufficient for tight binding, and represents the core binding element of the promoter. We propose a model in which part of the upstream binding energy is used by T7 RNA polymerase to melt the downstream initiation region of the promoter. We also show that the presence of magnesium is necessary for optimal binding, but not for specific enzyme-promoter complex formation, and we propose that magnesium is not required for melting of the promoter.

Thermodynamic and kinetic measurements of promoter binding by T7 RNA polymerase.

Previous steady state kinetic studies of the initiation of transcription by T7 RNA polymerase have shown that melting of the DNA helix near the transcription start site is not rate limiting [Maslak, M., & Martin, C. T. (1993) Biochemistry 32, 4281-4285]. In the current work, fluorescence changes in a nucleotide analog incorporated within the promoter are used to monitor changes in the DNA helix associated with polymerase binding. The fluorescence of 2-aminopurine has been previously shown to depend on the environment of the base, with fluorescence increasing in the transition from a double-stranded to a single-stranded environment [Xu, D., Evans, K.O., & Nordlund, T. M.(1994) Biochemistry 33, 9592-9599]. Fluorescence changes associated with polymerase binding to promoters incorporating 2-aminopurine at positions -4 through -1 support a model which includes melting, in the statically bound complex, of the region of the promoter near the start site. Equilibrium titrations at 25 degrees C with label at position -2 provide a thermodynamic measure of the dissociation constant (Kd = 4.8 nM) for promoter binding, while stopped-flow kinetic assays measure the apparent association (k1 = 5.6 x 10(7) M-1 s-1) and dissociation (k-1 = 0.20 s-1) rate constants for simple promoter binding (the ratio k-1/k1 = 3.6 nM, in good agreement with the thermodynamic measurement of Kd). These results suggest that binding is close to the diffusion-controlled limit and helix melting is extremely rapid. In studies of structurally altered promoters, a base functional group substitution at position -10 is shown to significantly decrease k1, with little effect on k-1. In contrast, removal of the nontemplate strand from position +1 downstream results in a large decrease in k-1, with no significant effect on k1.

Major groove recognition elements in the middle of the T7 RNA polymerase promoter.

T7 RNA polymerase recognizes a relatively small promoter extending only 17 base pairs upstream from the start site for transcription. A model for this recognition suggests that the enzyme interacts with the major groove of duplex DNA in the region centered at position -9 [Muller, D.K., et al. (1989) Biochemistry 28, 3306-3313], and recent kinetic analyses of promoters containing base analogs at positions -10 and -11 have provided support for this model [Schick, C., & Martin, C.T. (1993) Biochemistry 32, 4275-4280; Schick, C., & Martin, C.T. (1995) Biochemistry 34, 666-672]. In the current work, we extend this analysis across the proposed major groove, identifying specific base functional group contacts at positions -9 through -5. Specifically, the 6-carbonyl of guanine at positions -9 and -7, the 6-amino group of adenine at position -8, the 5-methyl group of thymine at position -6 and the 2-amino group of guanine at position -5 are identified as primary contacts. The results strongly support the model for duplex recognition in this region of the promoter and suggest that recognition continues along one face of the helix beyond the major groove and into the adjoining minor groove at position -5, where helix melting begins.

Identification of essential amino acids within the proposed CuA binding site in subunit II of Cytochrome c oxidase.

To explore the nature of proposed ligands to the CuA center in cytochrome c oxidase, site-directed mutagenesis has been initiated in subunit II of the enzyme. Mutations were introduced into the mitochondrial gene from the yeast Saccharomyces cerevisiae by high velocity microprojectile bombardment. A variety of single amino acid substitutions at each of the proposed cysteine and histidine ligands (His-161, Cys-196, Cys-200, and His-204 in the bovine numbering scheme), as well as at the conserved Met-207, all result in yeast which fails to grow on ethanol/glycerol medium. Similarly, all possible paired exchange Cys,His and Cys,Met mutants show the same phenotype. Furthermore, protein stability is severely reduced as evidenced by both the absence of an absorbance maximum at 600 nm in the spectra of mutant cells and the underaccumulation of subunit II, as observed by immunolabeling of mitochondrial extracts. In the same area of the protein, a variety of amino acid substitutions at one of the carboxylates previously implicated in binding cytochrome c, Glu-198, allow (reduced) growth on ethanol/glycerol medium, with normal intracellular levels of protein. These results suggest that a precise folding environment of the CuA site within subunit II is essential for assembly or stable accumulation of cytochrome c oxidase in yeast.

Tests of a model of specific contacts in T7 RNA polymerase-promoter interactions.

The T7, T3, and SP6 RNA polymerases represent a highly homologous family of enzymes that recognize similarly homologous promoter DNA sequences. Despite these similarities, the enzymes are highly specific for their respective promoters. Studies of mutant RNA polymerases have linked a specific amino acid residue in the protein to recognition of bases at positions -11 and -10 in the promoter [Raskin, C. A., et al. (1992) J. Mol. Biol. 228, 506-515]. In kinetic analyses of transcription from synthetic promoters containing base-analog substitutions, we have recently shown that at positions -11 and -10 of the T3 promoter, T3 RNA polymerase recognizes functional groups along the nontemplate strand wall of the major groove [Schick, C., & Martin, C. T. (1993) Biochemistry 32, 4275-4780]. We now extend these studies to the homologous region of the T7 promoter. The results confirm extrapolations from the T3 system and show that T7 RNA polymerase recognizes corresponding functional groups at positions -11 and -10 of the T7 promoter. The results are consistent with a direct readout model for recognition of these bases [Raskin, C. A., et al. (1992) J. Mol. Biol., 228, 506-515], in which the 6-carbonyl and 7-imino groups of the nontemplate guanine at position -11 and the 6-amino group of the nontemplate adenine at position -10 of the T7 promoter are directly involved in binding. The results further support an overall model for promoter recognition in which the enzyme binds to one face of the duplex DNA in this upstream region of the promoter.

Effects of solution conditions on the steady-state kinetics of initiation of transcription by T7 RNA polymerase.

The T7 family of DNA-dependent RNA polymerases presents an ideal model system for the study of fundamental aspects of transcription. The small size of the promoter allows a variety of studies based on simple steady-state kinetics in the synthesis of a five-base runoff transcript. This assay can be used to characterize the effects on the initiation of transcription of simple modifications to potential protein or DNA specificity contacts. In the current work, in vitro conditions for this assay have been identified which optimize the apparent Km for the interaction between the enzyme and the promoter DNA. The addition to the reaction mixture of 0.05% Tween-20 and the substitution of 10 mM NaCl by 100 mM potassium glutamate not only improves the quality of the kinetic assays but also decreases Km by about an order of magnitude (strengthening the interaction between polymerase and its promoter). As observed for DNA binding in other systems, the parameter Km increases substantially with increasing [NaCl], but the salt dependence is shifted to higher concentrations as a function of [KGlu]. Thermal denaturation of the protein, monitored by circular dichroism spectroscopy, confirms the effects of salt and supports a model in which Cl- and other anions compete for phosphate binding sites on the protein. Finally, while Km is highly dependent on [NaCl], the measured kcat is relatively insensitive to salt. These data indicate that the parameters Km and kcat reflect changes respectively in promoter binding and in a rate-limiting step or steps leading to the initiation of transcription.

Tests of a model for promoter recognition by T7 RNA polymerase: thymine methyl group contacts.

The DNA-dependent RNA polymerase from bacteriophage T7 is highly specific for a 17 base promoter sequence. Interactions between T7 RNA polymerase and its promoter DNA have been probed using modified oligonucleotides and a steady-state kinetic assay. The incorporation of deoxyuridine in place of thymidine at individual sites in the promoter sequence results in the replacement of an exocyclic methyl group by hydrogen (effectively removing the thymine methyl). This substitution has been placed individually at each of the thymines in the T7 consensus promoter. Many of these substitutions do not affect binding or catalysis; however, the thymine methyl group at position-6 is critical to recognition. The kinetic parameter Km increases approximately 10-fold while kcat is only slightly affected, suggesting that this thymine methyl is critical to binding specificity, but not to the kinetics of initiation. Two methyl groups near the start site on the template strand (at positions -1 and -3) also contribute to promoter specificity, while nearby methyl groups on the nontemplate strand do not. The implications of these results are discussed with respect to recent models for promoter binding.

Identification of specific contacts in T3 RNA polymerase-promoter interactions: kinetic analysis using small synthetic promoters.

The T7, T3, and SP6 RNA polymerases recognize very similar, yet distinct, promoter sequences. The high homology among the promoter sequences suggests that differential promoter recognition must derive from relatively small changes in the protein. Steady-state kinetic analyses of transcription from the T3 consensus promoter and from promoters modified in the region critical to specific recognition reveal details concerning which functional groups contribute to this recognition. Modifications include base pair substitutions, single base substitutions (mismatches), and simple functional group modifications at unique sites in the promoter. The results show that T3 RNA polymerase recognizes the amino group on the nontemplate cytidine in the major groove at position -10, while the identity of the base on the template strand is less critical to binding. In contrast, recognition at position -11 allows a greater range of modifications and seems to have a more complex recognition. The results do not seem to be consistent with a single recognition contact at this position; however, some groups may be ruled out as simple recognition contacts. While major groove modifications weaken binding at positions -10 and -11, the removal of an exocyclic amino group from the minor groove at either position does not disrupt binding, further supporting a model for promoter recognition in which the enzyme binds to one face of closed duplex DNA in this region. The effects of these changes in the DNA structure on the kinetics of initiation are compared to complementary results from the T7 system.

Kinetic analysis of T7 RNA polymerase transcription initiation from promoters containing single-stranded regions.

T7 RNA polymerase is highly specific for the initiation of transcription from a relatively small consensus promoter sequence. Previous footprinting studies suggested that the enzyme binds specifically to a fully closed duplex form of the promoter, recognizing functional groups along one face of the helix [Muller, D. K., Martin, C. T., & Coleman, J. E. (1989) Biochemistry 28, 3306-3313]. Steady-state kinetic analysis of oligonucleotide-based promoters shows that removal of the nontemplate strand completely within the message region of the DNA (positions +1 through +5) results in no change in binding (as reflected in the parameter Km) and a 2-fold increase in kinetics (as reflected in kcat). Further deletion of the nontemplate strand as far upstream as position -4 has no effect on binding, and although deletion upstream through position -6 weakens binding, specific initiation continues at a high rate. The temperature dependence of the initiation kinetics shows a single apparent activation energy of approximately 26 kcal/mol for the fully duplex promoter. Similar measurements on the promoter lacking the nontemplate strand in the message region show that less than 10% of this barrier is related to melting of the downstream region of the promoter. These results lead us to revise the previous model for recognition to include specific binding to a form of the promoter which is duplex upstream of about position -6 and melted downstream through the start site. Within the melted region, the polymerase interacts significantly only with the template strand of the promoter DNA.

Reaction of single-stranded DNA with hydroxyl radical generated by iron(II)-ethylenediaminetetraacetic acid.

This study demonstrates that the reaction of Fe(II)-EDTA and hydrogen peroxide with the single-stranded nucleic acids d(pT)70 and a 29-base sequence containing a mixture of bases results in substantial damage which is not directly detected by gel electrophoresis. Cleavage of the DNA sugar backbone is enhanced significantly after the samples are incubated at 90 degrees C in the presence of piperidine. The latter reaction is used in traditional Maxam-Gilbert DNA sequencing to detect base damage, and the current results are consistent with reaction of the hydroxyl radical with the bases in single-stranded DNA (although reaction with sugar may also produce adducts that are uncleaved but labile to cleavage by piperidine). We propose that hydroxyl radicals may react preferentially with the nucleic acid bases in ssDNA and that reaction of the sugars in dsDNA is dominant because the bases are sequestered within the double helix. These results have implications both for the study of single-stranded DNA binding protein binding sites and for the interpretation of experiments using the hydroxyl radical to probe DNA structure or to footprint double-stranded DNA binding protein binding sites.

T7 RNA polymerase interacts with its promoter from one side of the DNA helix.

The interactions of T7 RNA polymerase with its promoter DNA have been previously probed in footprinting experiments with either DNase I or (methidiumpropyl-EDTA)-Fe(II) to cleave unprotected DNA [Basu, S., & Maitra, U. (1986) J. Mol. Biol. 190, 425-437. Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. Both of these reagents have drawbacks; DNase I is a bulky reagent and so provides low resolution, and (methidiumpropyl-EDTA)-Fe(II) intercalates into DNA and is therefore biased toward cleavage of double-stranded DNA. In this study, the interaction between the polymerase and the promoter has been probed with Fe(II)-EDTA. This reagent generates reactive hydroxyl radicals free in solution, which produces a more detailed picture of the polymerase-promoter complex. Two protected regions are observed on each of the two promoter DNA strands: from position -17 to position -13 and from position -7 to position -1 on the coding strand and from position -14 to position -9 and from position -3 to position +2 on the noncoding strand. From this pattern it is clear that if recognition occurs via double-stranded B-form DNA, then the protected regions lie on one face of the DNA helix, and therefore the enzyme must interact predominantly from one side of the DNA helix. Digestion of the DNA in a polymerase-promoter complex with a single-strand-specific endonuclease shows that a small region of the noncoding strand near position -5 is susceptible to cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

T7 RNA polymerase does not interact with the 5'-phosphate of the initiating nucleotide.

The study of transcription kinetics by T7 RNA polymerase is facilitated by the small size of its promoter, allowing the use of synthetic oligonucleotide templates with carefully defined sequences. We have previously used this approach to measure Michaelis-Menten steady-state kinetics for production of the five-base runoff transcript GGACU. In particular, Km for the interaction between enzyme and template under saturating levels of all four nucleotide triphosphates was shown to be approximately 0.02 microM. We now show that the corresponding Km and Vmax for initiation on a similar template coding for the runoff transcript GACU are the same as for the earlier study (Km = 0.02 microM; kcat = 40-50 min-1). This new template allows the measurement Km for association of the initial nucleotide GTP with enzyme or with the enzyme-DNA complex. The results show that KGTPm (0.60 mM) is somewhat higher than earlier approximations of Km for addition of elongating GTP during the later phase of processive elongation. As expected, the (initiating) Km for the GTP analogue ITP (KITPm) is increased (by about 2-fold), presumably as a result of weakened Watson-Crick base pairing. However, comparison of Km values for the GTP analogues GMP and guanosine shows little effect on substitution of the 5'-triphosphate by monophosphate or by a hydroxyl, respectively. This result suggests that a single active site has been evolutionarily adapted to accept from the 5' end of a waiting nucleotide both a 5'-triphosphate at initiation and a 5'-monophosphate ester (RNA) during elongation.(ABSTRACT TRUNCATED AT 250 WORDS)