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PubMed1985

On the coordination properties of Eu3+ bound to tRNA.

The luminescence properties of Eu3+ have been used to investigate the binding and coordination properties of the ion with tRNA, as an attempt to resolve the discussion of whether metal ions bind to tRNA in solution only by Debye-Hückel screening, or whether direct coordination to specific sites may occur. Binding studies with Escherichia coli tRNAmet/f (taking advantage of 4-thiouracil-sensitized Eu3+ emission) distinguish three classes of binding affinities. Two of these are single sites with affinities approx. 10(4) and approx. 10(3) tighter than the nonspecific affinity of Eu3+ for native DNA. Mg2+ competes for binding at both these sites. Measurement of the lifetime and excitation spectrum of Eu3+ bound to the highest affinity site shows that the ion has two to five non-phosphate ligands in its inner coordination sphere. The existence of this coordinated site demonstrates that electrostatic screening is not the only mechanism for metal ion interaction with tRNA. The coordination properties of the high-affinity Eu3+ site do not agree with the properties of any of the metal ion sites found in the two tRNAphe crystal forms. Possible reasons for this discrepancy are discussed; it may be that ions bind differently to isolated molecules in solution than to molecules packed in a crystal lattice.

Binding Sites↗

PubMed2000

ZrIV-tetraphenylporphyrinates as nuclease mimics: structural, kinetic and mechanistic studies on phosphate diester transesterification.

The Zr(IV)-tetraphenylpor-phyrinates Zr(TPP)(X,X'), (X,X' = -OAc, -OMe, Cl ) 4-6, 8 were prepared and their complexing properties as well as catalytic properties towards solvolysis of the phosphate diesters hpp (2), dmp (3) and pmp (16) characterised. The diesters 2 and 16, representing model phosphates for RNA and DNA, were substrates for the catalyst Zr(TPP)Cl2 (4), and rate accelerations over background by 6-9 orders of magnitude were measured. These accelerations are comparable to those of dinuclear transition metal catalysts and lanthanide ions. Catalytic turnover was observed. Kinetic studies revealed that the catalytically active species of 4 in the solvolysis of 2 and 16 in methanol-containing solvents are dinuclear complexes containing either one or two phosphate esters depending upon the phosphate concentration. Besides the usual solvolysis pathway of the RNA model hpp (2), which proceeds via the cyclophosphate 20, a second, unusual pathway via direct substitution of the hydroxypropyl substituent was found. X-ray analysis of the Zr(TPP)(dmp) complex 19 revealed a dinuclear structure with two bridging dmp ligands and one monomethyl phosphate unit. In 19 one of the two dmp residues occurs in a very unusual high energy ac,ap conformation. Based on this structure and on the kinetic data, mechanistic models for the two solvolysis reaction pathways were developed. From an extensive CSD search on phosphodiester structures no correlation between P-O ester bond lengths and diester conformations could be found. However, P-O ester bonds decrease in length with increasing formal charge of the complexing metal ions. This underlines the higher importance of electrostatic activation relative to stereoelectronic effects in phosphodiester hydrolysis.

Catalysis↗

PubMed1986

Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization.

A sensitive membrane filter assay has been used to examine the kinetic and equilibrium properties of the interactions between Escherichia coli ribosomal protein S8 and 16S rRNA. In standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 0.35 M KCl) the apparent association constant is 5 +/- 0.5 X 10(-7) M-1. The interaction is highly specific, and the kinetics of the reaction are consistent with the apparent association constant. Nevertheless, the rate of association is somewhat slower than that expected for a diffusion-controlled reaction, suggesting some steric constraint. The association is only slightly affected by temperature (delta H = -1.8 kcal/mol). The entropy change [delta S = +29 cal/(mol K)] is clearly the main driving force for the reaction. The salt dependence of Ka reveals that five ions are released upon binding at pH 7.5 and in the presence of 10 mM magnesium. The substitution of various anions for Cl- has an appreciable effect on the magnitude of Ka, following the order CH3COO- greater than Cl- greater than Br-, thus indicating the existence of anion binding site(s) on S8. An equal number of ions were released when Cl- was replaced by CH3COO-, but the absence of anion release upon binding cannot be excluded. On the other hand, the free energy of binding appears not to be exclusively electrostatic in nature. The effect of pH on both temperature and ionic strength dependence of Ka has been examined. It appears that protonation of residue(s) (with pK congruent to 9) increases the affinity via a generalized charge effect. On the other hand, deprotonation of some residue(s) with a pK congruent to 5-6 seems to be required for binding. Furthermore, the unique cysteine present in S8 was shown to be essential for binding.

Escherichia coli↗

PubMed2003

Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovani.

The amino acid sequences of the C-terminal domain (CTD) of the type II DNA topoisomerases are divergent and species specific as compared with the highly conserved N-terminal and central domains. A set of C-terminal deletion mutants of Leishmania donovani topoisomerase II was constructed. Removal of more than 178 amino acids out of 1236 amino acid residues from the C-terminus inactivates the enzyme, whereas removal of 118 amino acids or less has no apparent effect on the ability of the parasite enzyme to complement a temperature-sensitive mutation of the Saccharomyces cerevisiae topoisomerase II gene. Deletion analysis revealed a potent nuclear localization signal (NLS) within the amino acid residues 998-1058. Immunomicroscopy results suggest that the removal of an NLS in the CTD is likely to contribute to the physiological dysfunction of these proteins. Modeling of the LdTOP2 based on the crystal structure of the yeast type II DNA topoisomerase showed that the parasite protein assumes a structure similar to its yeast counterpart harboring all the conserved residues in a structurally similar position. However, a marked difference in electrostatic potential was found in a span of 60 amino acid residues (998-1058), which also do not have any homology with topoisomerase II sequences. Such significant differences can be exploited by the structure-based design of selective inhibitors using the structure of the Leishmania enzyme as a template.

Amino Acid Sequence↗

PubMed1981

Interaction of the ruthenium red cation with nucleic acid double helices.

The hexapositive complex cation ruthenium red very effectively stabilizes DNA and RNA double helices against thermal denaturation. In the presence of nucleic acid helices, this symmetric cation acquires an extrinsic CD spectrum near the wavelength of the dye's maximum absorbance. Competition experiments with single-stranded polyd(T) show this induced CD to be the result of selective binding to helical sites. The preferential affinity of ruthenium red for double helical binding sites is so great that it brings about biphasic absorbance- temperature profiles of polyd(A-T) at low [cation]: [polynucleotide phosphate]. The visible CD signal and fraction of helix melting at the upper transition increases with ruthenium red concentration until approximate charge neutrality is reached. These interactions, which have been studied in detail with the poly(U-U) helix as well as polyd(A-T), are likely largely electrostatic, since sufficient [NaCl] eliminates the bipliasic melting of polyd(A-T), renders the ultraviolet absorbance of poly (U) insensitive to ruthenium red, and abolishes the induced CD effects. The bipliasic melting of polyd(A-T) at intermediate [dye] is attributed to saturation of remaining double helical segments by cation migration from newly melted regions- Furthermore, virtually no change was observed in the induced CD upon melting through the first transition, whereas the effect is destroyed upon inciting through the second transition. A quantitative treatment of the data is used to obtain binding site size and association constant for the complex. The induced effect may prove useful in the exploration of exposed nucleic acid helical structure in such complex particles as nucleosomes or ribosomes.

Journal Article↗

PubMed2000

Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch.

Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of Thermus thermophilus RRF was determined at 2.6 A resolution. It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349-2352), the interdomain angle differs by 33 degrees in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing. The hinge connects hydrophobic junctions of domains 1 and 2. The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF. First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop. Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop. These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility. These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a "gooseneck" elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other. Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G. These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.

Alleles↗

PubMed2005

Two regions in the N-terminal domain of ionotropic glutamate receptor 3 form the subunit oligomerization interfaces that control subtype-specific receptor assembly.

The N-terminal domain (NTD) of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1-N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor delta2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.

Alanine↗

PubMed2004

Highly sensitive amperometric detection of genomic DNA in animal tissues.

A simple and highly sensitive method for the detection of genomic DNA in tissue samples is described. It is based on amperometric detection of target DNA by forming an analyte/polymeric activator bilayer on a gold electrode. The biotinylated target DNA is hybridized to oligonucleotide capture probes immobilized on the gold electrode, forming the first layer. A subsequent binding of glucose oxidase-avidin conjugate to the target DNA and the introduction of a second layer of a redox polymer to the electrode, via layer-by-layer electrostatic self-assembly, allow for electrochemical detection of the catalytic oxidation current of glucose in a PBS solution. Less than 2.0 fg of rat genomic DNA, for both regulated and house-keeping genes, can be easily detected in 2.5 microl droplets. The proposed procedure shows very high specificity for genomic DNA in a RT-PCR mixture.

Animals↗

PubMed1996

Role of the "helix clamp" in HIV-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis.

Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the "helix clamp" (Hermann, T., Meier, T., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of alpha H, alanine substitution in alpha I did not have a significant effect on template.primer (T.P) binding as revealed by a lack of an effect on Km, T.P, Ki for 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, koff, T.P and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of alpha I lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T.P (L282A and R284A) demonstrated that these mutant enzymes could bind T.P stoichiometrically and tightly. In contrast, active site concentrations determined from "burst" experiments suggest that the lower activity is due to a smaller populations of enzyme bound productively to T.P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of alpha H and the DNA minor groove, 3-5 nucleotides from the 3'-primer terminus, are suggested to be critical for DNA binding and fidelity.

Alanine↗

PubMed2004

Induction of cytochrome P4501A (CYP1A) by clotrimazole, a non-planar aromatic compound. Computational studies on structural features of clotrimazole and related imidazole derivatives.

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). High-affinity AhR ligands are planar polyaromatic molecules such as the prototypic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present work investigated the ability of the imidazole derivative, clotrimazole [1-(2'chlorotrityl)imidazole, CLO], to induce CYP1A in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes at the catalytic activity (determined as 7-ethoxyresorufin-O-deethylase, EROD) and at the transcriptional level. CLO resulted in a significant increase of hepatocyte EROD activity and CYP1A mRNA at a concentration of 1.56 microM. Computational studies on the molecular structure of CLO show that CLO is unlikely to take a planar conformation. Further indications that CLO does not behave like a planar AhR ligand come from the experimental observation that co-incubation of trout hepatocytes with CLO and the AhR antagonist, alpha-naphthoflavone (alpha-NF), did not result in an inhibition of CLO induction of CYP1A mRNA, whereas alpha-NF was able to inhibit CYP1A induction by the prototpyic, planar AhR ligand, beta-naphthoflavone. The experimental findings on CLO agree with previous results obtained for another non-planar imidazole derivative, 1-benzylimidazole (BIM). Further, computational studies showed that the non-planar imidazoles, BIM and CLO, are highly similar with respect to some electrostatic properties, namely the dipole moment and the molecular electrostatic potential (MEP). Overall our experimental and computational studies suggest that transcriptional activation of CYP1A by the imidazole derivatives CLO and BIM is mediated by a mechanism different to that of prototypic CYP1A inducers such as the planar AhR-ligands.

Animals↗

PubMed2005

Mutations in betaB3-crystallin associated with autosomal recessive cataract in two Pakistani families.

PURPOSE: To identify the disease locus for autosomal recessive congenital cataracts in consanguineous Pakistani families. METHODS: Two Pakistani families were ascertained, patients were examined, blood samples were collected, and DNA was isolated. A genome-wide scan was performed using >382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members. Two-point lod scores were calculated, haplotypes were formed by inspection, and candidate genes were sequenced. Real-time quantitative PCR techniques were used to determine the mRNA levels, and molecular modeling was performed to gain a better understanding of the significance of the disease-causing mutation. RESULTS: In the genome-wide scan, maximum lod scores of 2.67 and 2.77 for family 60004 and 2.02 and 2.04 for family 60006 were obtained for markers D22S539 and D22S315, respectively. The linked region, 22.7 cM (10 Mb) flanked by markers D22S420 and D22S1163, contains the beta-crystallin gene cluster including the genes CRYBA4, CRYBB1, CRYBB2, and CRYBB3. Sequencing of these genes showed a G-->C transition in exon 6 of CRYBB3 resulting in a p.G165R change in the betaB3-crystallin protein that cosegregates with the disease in both families. Real-time PCR analysis suggested that betaB3-crystallin mRNA levels approximate those of other betagamma-crystallins. Molecular modeling predicted changes in electrostatic potential that would be expected to reduce the stability of the fourth Greek-key motif, and hence the entire protein, dramatically. CONCLUSIONS: For the first time, a mutation in CRYBB3 is reported in two consanguineous Pakistani families with autosomal recessive congenital cataracts.

Amino Acid Sequence↗

PubMed2006

Protein transduction technology: a novel therapeutic perspective.

The direct intracellular delivery of proteins has, until recently, been difficult to achieve, due primarily to the bioavailability barrier of the plasma membrane. During the past 15 years, a variety of peptides called protein transduction domains (PTDs) or cell penetrating peptides (CPPs), have been characterized for their ability to translocate into live cells. The most commonly studied are homeodomain transcription factors such as Antennapedia, the herpes simplex virus (HSV) type 1 protein VP22, and the human immunodeficiency virus (HIV-1) transactivator TAT protein. Recently, polyarginine exhibits even greater efficiency in terms of delivery of several peptides and proteins. Numerous examples of biologically active full-length proteins and peptides have been delivered to cells and tissues, both in vitro and in vivo. These studies offer new avenues for treatment of several diseases. The main mechanism of protein transduction is an electrostatic interaction with the plasma membrane, penetration into cells by macropinocytosis, and a release to cytoplasm and nuclei by retrograde transport. Moreover, the intercellular transfer of endogenous transcription factors, such as TAT and homeoproteins, seems to point to an original and important mode of signal transduction. The protein transduction systems have opened up several possibilities, not only for the development of new peptide/protein drugs but also for consideration of their physiological and developmental implications.

Cell Membrane↗

PubMed2003

Long-chain acyl-CoA esters and phosphatidylinositol phosphates modulate ATP inhibition of KATP channels by the same mechanism.

Phosphatidylinositol phosphates (PIPs, e.g. PIP2) and long-chain acyl-CoA esters (e.g. oleoyl-CoA) are potent activators of KATP channels that are thought to link KATP channel activity to the cellular metabolism of PIPs and fatty acids. Here we show that the two types of lipid act by the same mechanism: oleoyl-CoA potently reduced the ATP sensitivity of cardiac (Kir6.2/SUR2A) and pancreatic (Kir6.2/SUR1) KATP channels in a way very similar to PIP2. Mutations (R54Q, R176A) in the C- and N-terminus of Kir6.2 that greatly reduced the PIP2 modulation of ATP sensitivity likewise reduced the modulation by oleoyl-CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl-CoA and PIP2 on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP4-, ADP3- and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP2-)) and diadenosine tetraphosphate (Ap4A5-) ruled out a mechanism where oleoyl-CoA or PIP2 attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl-CoA) did not activate but inhibited KATP channels (IC50 = 265 +/- 33 muM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap4A) are ligands of the inhibitory ATP-binding site on Kir6.2.

ATP-Binding Cassette Transporters↗

PubMed2006

Deuterium isotope effects in A:T and A:U base pairs: a computational NMR study.

Recent measurements of trans-hydrogen bond deuterium isotope effects (DIEs) on 13C chemical shifts in nucleic acids (Vakonakis, I.; LiWang, A. C. J. Biomol. NMR 2004, 29, 65; J. Am. Chem. Soc. 2004, 126, 5688) have led to intriguing results: (i) the DIEs of A:T pairs in DNA are about 5 ppb smaller than those of A:U in RNA and (ii) A:T DIEs vary by as much as 13 ppb among the oligonucleotides. The first observation suggests that inter-base H-bonds in RNA may be stronger than those in DNA, while the second indicates that the conformation of the base pair modulates the transmission of the isotope effect across the hydrogen bond. In an effort at providing a rationale--so far unknown--for the observed DIEs in nucleic acids, density functional theory and hybrid Car-Parrinello/molecular mechanical calculations of DIEs on nucleosides and nucleotides in the gas phase and in aqueous solution have been performed. The calculations suggest that (i) the DIE in an isolated A:T base pair differs from that in an A:U base pair because of the changes in the magnetic properties caused by the replacement of a methyl group on passing from U to T, (ii) the DIEs depend crucially on the conformation of the base pairs, and (iii) the DIEs are strongly affected by magnetic and electrostatic interactions with the surrounding environment.

Journal Article↗

PubMed2002

Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells.

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.

Animals↗

PubMed1993

Discrimination between transfer-RNAs by tyrosyl-tRNA synthetase.

We have constructed a model of the complex between tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and tRNA(Tyr) by successive cycles of predictions, mutagenesis of TyrRS and molecular modeling. We confront this model with data obtained independently, compare it to the crystal structures of other complexes and review recent data on the discrimination between tRNAs by TyrRS. Comparison of the crystal structures of TyrRS and GlnRS, both of which are class I synthetases, and comparison of the identity elements of tRNA(Tyr) and tRNA(Gln) indicate that the two synthetases bind their cognate tRNAs differently. The mutagenesis data on tRNA(Tyr) confirm the model of the TyrRS:tRNA(Tyr) complex on the following points. TyrRS approaches tRNA(Tyr) on the side of the variable loop. The bases of the first three pairs of the acceptor stem are not recognized. The presence of the NH2 group in position C6 and the absence of a bulky group in position C2 are important for the recognition of the discriminator base A73 by TyrRS, which is fully realized only in the transition state for the acyl transfer. The anticodon is the major identity element of tRNA(Tyr). We have set up an in vivo approach to study the effects of synthetase mutations on the discrimination between tRNAs. Using this approach, we have shown that residue Glu152 of TyrRS acts as a purely negative discriminant towards non-cognate tRNAs, by electrostatic and steric repulsions. The overproductions of the wild type TyrRSs from E coli and B stearothermophilus are toxic to E coli, due to the mischarging or the non-productive binding of tRNAs. The construction of a family of hybrids between the TyrRSs from E coli and B stearothermophilus has shown that their sequences and structures have remained locally compatible through evolution, for folding and function, in particular for the specific recognition and charging of tRNA(Tyr).

Binding Sites↗

PubMed1984

Protein-DNA recognition.

Several general principles emerge from the studies of Cro, lambda repressor, and CAP. The DNA-binding sites are recognized in a form similar to B-DNA. They do not form cruciforms or other novel DNA structures. There seem to be proteins that bind left-handed Z-DNA (87) and DNA in other conformations, but it remains to be seen how these structures are recognized or how proteins recognize specific sequences in single-stranded DNA. Cro, repressor, and CAP use symmetrically related subunits to interact with two-fold related sites in the operator sequences. Many other DNA-binding proteins are dimers or tetramers and their operator sequences have approximate two-fold symmetry. It seems likely that these proteins will, like Cro, repressor, and CAP, form symmetric complexes. However, there is no requirement for symmetry in protein-DNA interactions. Some sequence-specific DNA-binding proteins, like RNA polymerase, do not have symmetrically related subunits and do not bind to symmetric recognition sequences. Cro, repressor, and CAP use alpha-helices for many of the contacts between side chains and bases in the major groove. An adjacent alpha-helical region contacts the DNA backbone and may help to orient the "recognition" helices. This use of alpha-helical regions for DNA binding appears to be a common mode of recognition. Most of the contacts made by Cro, repressor, and CAP occur on one side of the double helix. However, lambda repressor contacts both sides of the double helix by using a flexible region of protein to wrap around the DNA. Recognition of specific base sequences involves hydrogen bonds and van der Waals interactions between side chains and the edges of base pairs. These specific interactions, together with backbone interactions and electrostatic interactions, stabilize the protein-DNA complexes. The current models for the complexes of Cro, repressor, and CAP with operator DNA are probably fundamentally correct, but it should be emphasized that model building alone, even when coupled with genetic and biochemical studies, cannot be expected to provide a completely reliable "high-resolution" view of the protein-DNA complex. For example, the use of standard B-DNA geometry for the operator is clearly an approximation.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Sequence-dependent DNA structure. The role of base stacking interactions.

The sequence-dependent structure of DNA is analysed on the basis of the energetics of the base stacking (pi-pi) interactions. The conformational preferences of the ten possible base-pair steps in double-helical DNA have been calculated and compared with experimental data from X-ray fibre diffraction, X-ray crystal structures and gel-running experiments. The calculations account for many features of sequence-dependent DNA structure, including polymorphism in DNA, the lack of polymorphism in RNA, the structure of Z-DNA, bistability in pyrimidine-purine (YR) steps, the origin of propeller twist and buckle and the role of TATA sequences at the sites of origin of replication. The computational model used specifically allows for the charge distribution associated with the out-of-plane pi-electron density of the bases. The results obtained are rationalized on the basis of the shapes and charge distributions of the bases. Calladine's cross-strand steric clashes at pyrimidine-purine (YR) and CX/XG steps are reproduced. In AX/XT steps, same-strand steric clashes occur between the thymine methyl group and the 5'-neighbouring sugar. They are the cause of the large negative propeller twist observed in A.T base-pairs. Steric clashes between the thymine methyl group and the 5'-neighbouring base block A-DNA conformations in AX/XT steps. Electrostatic interactions between partial atomic charges are most important for C.G base-pairs which are highly polarized. They lead to strong preferences for positive slide and negative slide conformations in CG and GC steps, respectively. This combination can be accommodated in poly(CG) by left-handed Z-DNA. Many of the more subtle sequence-dependent effects are caused by electrostatic interactions between the partial atomic charges on one base-pair and the out-of-plane pi-electron density on another. The effect is most important in CX-XG steps and leads to bistability. In general, electrostatic interactions cause non-zero slide. This is opposed by the hydrophobic effect which favours the zero slide B-type conformation. Thus, B-DNA is observed at high water content in fibres and electrostatic interactions force high or low slide A or C-DNA conformations at low water content. If two juxtaposed steps have very different conformational preferences, this incompatibility can lead to unusual structures such as Z-DNA or strain such as in TATA sequences.

Base Sequence↗