Specific binding sites for cations in bacteriorhodopsin.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn(2+) and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn(2+), which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be approximately 9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn(2+) bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.     

The binding sites for tRNA on eukaryotic ribosomes.

We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.     

Modified tRNAs for probing tRNA binding sites on the ribosome

Unusual chemical properties of hypermodified nucleosides N6-(threoninocarbonyl)adenosine (t6 A) located at position 37 and 3-(3-amino-3-carboxypropyl)uridine (acp3U) located at position 20:1 have been utilized for the introduction of photoreactive azidonitrophenyl probes to the anticodon loop and to the dihydrouridine loop of yeast tRNA(mMet) and lupin tRNA(mMet), respectively. The very efficient and selective modification procedures involve condensat on of the carboxyl group of t6A with ethylenediamine in the presence of a water soluble carbodiimide followed by acylation of the newly introduced amino group with the respective N-hydroxysuccinimide ester, and acylation of the primary amino or up of acp3U with the respective N-hydroxysuccinimide ester. Binding and crosslinking of the modified, uncharged tRNAs to E coli ribosome have been studied in the presence and absence of poly(AUG) as a message. Both tRNAs carrying about 20 A long photoreactive probes retain their binding activity and upon irradiation with visible light crosslink to the ribosome with high yields showing their usefulness for structural studies on the tRNA-ribosome complex.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

tRNA binding sites on the subunits of Escherichia coli ribosomes

Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity. Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA. Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent. The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies. Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site). Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes. In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA. Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site. The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes. 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits. The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.     

Affinities of tRNA binding sites of ribosomes from Escherichia coli

The binding affinities of tRNAPhe, Phe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E. coli 70S ribosomes were determined at various ionic conditions. For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used. Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites. The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 10(8) M-1 and 5 X 10(9) M-1, respectively. Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration. Phe-tRNAPhe binds to the A site with a similar affinity. Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 10(4). Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore. The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site. In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength. Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficient codon-anticodon interaction in the E site. On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site. Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation.     

Invasion of strongly binding oligonucleotides into tRNA structure

Interaction of yeast tRNA(Phe) with oligodeoxyribonucleotides containing 5-methylcytosine, 2-aminoadenine, and 5-propynyl-2'-deoxyuridine was investigated. The modified oligonucleotides show increased binding capacity although the association rates are similar for the modified and natural oligonucleotides. The most pronounced increase in association constant (70 times) due to the incorporation of the strongly binding units was achieved in the case of oligonucleotide complementary to the sequence 65-76 of the tRNA(Phe).     

Number of tRNA binding sites on 80 S ribosomes and their subunits

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5-20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA.     

Microarrays for identifying binding sites and probing structure of RNAs

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.     

The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.     

Interaction between alginates and manganese cations: identification of preferred cation binding sites

Algal and bacterial alginates have been studied by means of 13C NMR spectroscopy in presence of paramagnetic manganese ions in order to reveal the nature of their interaction with bivalent cations. It is found that the mannuronate blocks bind manganese cations externally near their carboxylate groups, while guluronate blocks show the capability to integrate Mn2+ into pocket-like structures formed by adjacent guluronate residues. In alternating mannuronate-guluronate blocks, manganese ions preferentially locate in a concave structure formed by guluronate-mannuronate pairs. Partial acetylation of the alginate generally reduces its capability to interact with bivalent cations, however, the selectivity of the binding geometry is conserved. The results may serve as a hint for the better understanding of the alginate gelation in presence of calcium ions.     

Chemical modification of low-density lipoprotein enhances the number of binding sites for divalent cations.

The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations.     

Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations.

Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 +/- 0.3 nm, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 +/- 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17-27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 +/- 2.0 nm, which decreased to 4.9 +/- 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.     

Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin

The binding of cations to ATP-G-actin has been assessed by measuring the kinetics of the increase in fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Ca2+ and Mg2+ compete for a single high-affinity site on ATP-G-actin with KD values of 1.5-15 nM for Ca2+ and 0.1-1 microM for Mg2+, i.e. with affinities 3-4 orders of magnitude higher than previously reported (Frieden, C., Lieberman, D., and Gilbert, H. R. (1980) J. Biol. Chem. 255, 8991-8993). As proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886), the Mg-actin complex undergoes a slow isomerization (Kis = 0.03-0.1) to a higher affinity state (K'D = 4-40 nM). The replacement of Ca2+ by Mg2+ at this high-affinity site causes a slow 10% increase in fluorescence that is 90% complete in about 200 s at saturating concentrations of Mg2+. Independently, Ca2+, Mg2+, and K+ bind to low-affinity sites (KD values of 0.15 mM for Ca2+ and Mg2+ and 10 mM for K+) which causes a rapid 6-8% increase in fluorescence (complete in less than 5 s). We propose that the activation step that converts Ca-G-actin to a polymerizable species upon addition of Mg2+ is the binding of Mg2+ to the low-affinity sites and not the replacement of Ca2+ by Mg2+ at the high-affinity site.     

Yeast seryl tRNA synthetase: interactions between the ATP binding site and the sites for tRNASer and L-serine.

T1 ribonuclease digestion of yeast tRNASer in the presence of seryl tRNA synthetase was used for monitoring the relationship between the substrate binding sites on the synthetase. It was found that (a) ATP displaces the tRNA from the synthetase with an effector affinity constant corresponding to the Km for ATP of 10 micron; (b) AMP and a number of nucleoside triphosphates, while influencing the rate of aminoacylation, do not displace the tRNA from the enzyme; (c) ADP and PPi inhibit the aminoacylation and the binding of tRNASer; (d) adenylyl diphosphonate is bound to the synthetase and lowers the protection of the tRNA against the nuclease attack in a similar way as does ATP; (e) interactions between the sites of L-serine and tRNASer could only be shown when both sites for serine were saturated and, in addition, the ATP analog or ADP was present. It is concluded that in seryl tRNA synthetase binding sites for ATP interact with the ones for tRNA as well as with the ones for serine. These findings contribute to the understanding of the mechanism of aminoacylation.