Phosphorylation of double-stranded DNAs by T4 polynucleotide kinase.

The phosphorylation by T4 polynucleotide kinase of various double-stranded DNAs containing defined 5'-hydroxyl end group structures has been studied. Particular emphasis was placed on finding conditions that allow complete phosphorylation. The DNAs employed were homodeoxyoligonucleotides annealed on the corresponding homopolymers, DNA duplexes corresponding to parts of the genes for alanine yeast tRNA, and a suppressor tyrosine tRNA from Escherichia coli. The rate of phosphoylation of DNAs with 5'-hydroxyl groups in gaps was approximately ten times slower than for the corresponding single-stranded DNA. At low concentrations of ATP, 1 muM, incomplete phosphorylation was obtained, whereas with higher concentrations of ATP, 30 muM, complete phosphorylation was achieved. In the case of DNAs with 5'-hydroxyl groups at nicks approximately 30% phosphorylation could be detected using 30 muM ATP. A DNA containing protruding 5'-hydroxyl group ends was phosphorylated to completion using the same conditions as for single-stranded DNA, i.e., a ratio between the concentrations of ATP and 5'-hydroxyl groups of 5:1 and a concentration of ATP of approximately 1 muM. For a number of DNAs containing protruding 3'-hydroxyl group ends and one DNA containing even ends incomplete phosphorylation was found under similar conditions. For all these DNAs a plateau level was observed varying from 20 to 45% of complete phosphorylation. At 20 muM and higher ATP concentrations, the phosphorylation was complete also for these DNAs. With low concentrations of ATP a rapid production of inorganic phosphate was noted for all the latter DNAs. The apparent equilibrium constants for the forward and reverse reaction were determined for a number of different DNAs, and these data revealed that the plateau levels of phosphorylation obtained at low concentrations of ATP for DNAs with protruding 3'-hydroxyl group and even ends is not a true equilibrium resulting from the forward and reverse reaction. It is suggested that the plateau levels are due to formation of inactive enzyme-substrate and enzyme-product complexes. For all double-stranded DNAs tested, except DNAs containing protruding 5'-hydroxyl group ends, addition of KCl to the reaction mixture resulted in a drastic decrease in the rate of phosphorylation, as well as in the maximum level phosphorylated. Spermine, on the other hand, had little influence. Both of these agents have previously been shown to activate T4 polynucleotide kinase using single-stranded DNAs as substrates (Lillehaug, J.R., and Kleppe, K. (1975), Biochemistry 14, 1221). The inhibition of phosphorylation of double-stranded DNAs by salt might be the result of stabilization of the 5'-hydroxyl group regions of these DNAs.     

Reinvestigation of phosphorylation of tRNA in Escherichia coli.

This report shows the results of the reinvestigation of tRNA phosphorylation in E. coli. The phosphorylation did not occur on suppressor seryl-tRNA but occurred on other tRNA species. The activity of tRNA phosphorylation was found in E. coli extracts and partially purified. On DEAE-Sephadex A50 and PAGE gel, the phosphorylated-tRNA showed a pattern different from that the natural suppressor serine tRNA.     

The binding of polyamines and of ethidium bromide to tRNA.

The binding of spermidine and ethidium bromide to mixed tRNA and phenylalanine tRNA has been studied under equilibrium conditions. The numbers and classes of binding sites obtained have been compared to those found in complexes isolated by gel filtration a low ionic strength. The latter complexes contain 10-11 moles of either spermidine or ethidium per mole of tRNA; either cation is completely displaceable by the other. In ethidium complexes, the first 2-3 moles are bound in fluorescent binding sites; the remaining 7-8 molecules bind in non-fluorescent form. At least one of the binding sites for spermidine appears similar to a binding site for fluorescent ethidium. Similar results are found with E. coli formylmethionine tRNA. Spermine, in excess of 18-20 moles per mole tRNA, causes precipitation of the complex. Putrescine does not form isolable complexes with yeast tRNA and displaces ethidium less readily from preformed ethidium-tRNA complexes. Under equilibrium conditions, in the absence of Mg++, there are 16-17 moles of spermidine bound per mole of tRNA as determined by equilibrium dialysis. Of these, 2-3 bind with a Ksence of 9 mM Mg++, the total number of binding sites is decreased slightly and there appears to be only one class of sites with a Ka = 600 M(-1). Quantitatively similar results are obtained for the binding of spermidine to yeast phenylalanine tRNA. When the interaction between ethidium bromide and mixed tRNA is studied by equilibrium dialysis or spectrophotometric titration, two classes of binding sites are obtained: 2-3 molecules bind with an average Ka = 6.6 x 10(5) M(-1) and 14-15 molecules bind with an average Ka = 4.1 x 10(4) M(-1). Spermidine, spermine, and Mg++ compete effectively for both classes of ethidium sites and have the effect of reducing the apparent binding constants for ethidium. When the binding of ethidium is studied by fluorometry, there are 3-4 highly fluorescent sites per tRNA. These sites are also affected by spermidine, spermine and Mg++. Putrescine has little effect on any of the classes of binding sites. These data are consistent with those found under non-equilibrium conditions. They suggest that polyamines bind to fairly specific regions of tRNA and may be involved in the maintenance of certain structural features of tRNA.     

Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase

The phosphorylation of sarcoplasmic reticulum ATPase with Pi in the absence of Ca2+ was studied by equilibrium and kinetic experimentation. The combination of these measurements was then subjected to analysis without assumptions on the stoichiometry of the reactive sites. The analysis indicates that the species undergoing covalent interaction is the tertiary complex E X Pi X Mg formed by independent interaction of the two ligands with the enzyme. The binding constant of Pi or Mg2+ to either free or partially associated enzyme is approximately equal to 10(2) M-1, and no significant synergistic effect is produced by one ligand on the binding of the other; the equilibrium constant (Keq) for the covalent reaction E X Pi X Mg E-P X Mg is approximately equal to 16, with kphosph = 53 s-1, and khyd = 3-4 s-1 (25 degrees C, pH 6.0, no K+). The phosphorylation reaction of sarcoplasmic reticulum ATPase with Pi is highly H+ dependent. Such a pH dependence involves the affinity of enzyme for different ionization states of Pi, as well as protonation of two protein residues per enzyme unit in order to obtain optimal phosphorylation. The experimental data can then be fitted satisfactorily assuming pK values of 5.7 and 8.5 for the two residues in the nonphosphorylated enzyme (changing to 7.7 for one of the two residues, following phosphorylation) and values of 50.0 and 0.58 for the equilibrium constants of the H2(E X HPO4) in equilibrium with H(E-PO3) + H2O and H(E X HPO4) in equilibrium with E-PO3 + H2O reactions, respectively. In addition to the interdependence of H+ and phosphorylation sites, an interdependence of Ca2+ and phosphorylation sites is revealed by total inhibition of the Pi reaction when two high affinity calcium sites per enzyme unit are occupied by calcium. Conversely, occupancy of the phosphate site by vanadate (a stable transition state analogue of phosphate) inhibits high affinity calcium binding. The known binding competition between the two cations and their opposite effects on the phosphorylation reaction suggest that interdependence of phosphorylation site, H+ sites, and Ca2+ sites is a basic mechanistic feature of enzyme catalysis and cation transport.     

Conformational states of yeast tRNA Phe in the complex with cognate and non cognate synthetases.

The influence of phenylalanyl-tRNA synthetase and seryl-tRNA synthetase on the conformation and structural kinetics of yeast tRNA Phe was investigated. Ethidium substituted for dihydrouracil at position 16 or 17 was used as a structural probe, showing the existence of three conformational states in tRNA. The distribution of states (T1, T2, T3) is changed only by the cognate synthetase towards T3 which probably is related to the X-ray structure. The binding of phenylalanyl-tRNA synthetase leads to an about 10-fold increase in the fast transition T1 in equilibrium or formed from T2 which has been assigned to changes in the anticodon loop conformation and to a 2-3 fold increase in the slow transition which probably extends to other parts of the tRNA molecule. The observed rates for the transition T2 in equilibrium or formed from T3 are close to that observed for the transfer of the activated phenylalanine to tRNA Phe. This raises the possibility that the conformational transition in tRNA is the rate limiting step in the charging reaction.     

Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate-aminoacyl-tRNA complexes

A new method for measuring the dissociation rate of the Escherichia coli elongation factor Tu-GTP--aminoacyl-tRNA complex has been developed and applied to the determination of the dissociation rates of ternary complexes formed between E. coli EF-Tu-GTP and a set of E. coli aminoacyl-tRNAs. The set of aminoacyl-tRNAs includes at least one tRNA coding for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. The results reveal that the dissociation rates vary for each ternary complex. Tu-GTP-Gln-tRNA dissociates the slowest and Tu-GTP-Val-tRNA the fastest of all noninitiator ternary complexes at 4 degrees C, pH 7.4. The equilibrium dissociation constant for Tu-GTP-Thr-tRNA has been determined to be 1.3 (0.4) X 10(-9) M under identical reaction conditions, and the absolute value of the equilibrium dissociation constant has been calculated for 28 ternary complexes from the relative equilibrium dissociation constant ratios previously measured [Louie, A., Ribeiro, N. S., Reid, B. R., & Jurnak, F. (1984) J. Biol. Chem. 259, 5010-5016]. The association rate of each ternary complex has been estimated from the ratio of the dissociation rate relative to the equilibrium dissociation constant. Tu-GTP-His-tRNA associates the fastest and Tu-GTP-Leu-tRNA1Leu the slowest. By inclusion of Tu-GTP-Met-tRNAfMet in the studies, evidence has been obtained that suggests that the initiator ternary complex does not function in the elongation cycle because the dissociation rate of the complex is very fast.     

Human tyrosine tRNA is also internally cleavable by E. coli ribonuclease P RNA ribozyme in vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA(Ala), tRNA(His), and tRNA(iMet)) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool detect destablized tRNA molecules from any species.     

Rapid solid phase isolation of 20 specific tRNAs from Escherichia coli.

A solid phase procedure has been developed for the rapid isolation of all 20 species of tRNA from Escherichia coli. The overall yields for a single preparation cycle ranged from 62 to 96%, the average being 80%. The values for the amino acid acceptor activities of the tRNA species equaled those reported in the literature for highly purified tRNAs. Starting from crude tRNA, a given tRNA species can easily be isolated in less than 2 h. One milliliter of the resin, which is reusable, is sufficient for the isolation of 200 mg of a specific tRNA. The procedure requires a bifunctional reagent, one moiety of which (--SO2Cl) reacts with the amino acid on the aminoacylated tRNA, the other, with the --SH group on the resin. Thus, only the desired tRNA species is bound to the resin; any of the other tRNAs in the filtrate can be isolated in another cycle. Raising the pH results in deacylation and release from the resin of the desired tRNA species. For tRNA Cys, it is necessary to block the --SH of cysteine prior to reaction with the bifunctional reagent. Side reactions involving the bifunctional reagent. Side reactions involving the bifunctional reagent and tRNA are either easily reversible or negligible (less than 0.01%).     

Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.     

Control of transfer RNA maturation by phosphorylation of the human La antigen on serine 366

Conversion of a nascent precursor tRNA to a mature functional species is a multipartite process that involves the sequential actions of several processing and modifying enzymes. La is the first protein to interact with pre-tRNAs in eukaryotes. An opal suppressor tRNA served as a functional probe to examine the activities of yeast and human (h)La proteins in this process in fission yeast. An RNA recognition motif and Walker motif in the metazoan-specific C-terminal domain (CTD) of hLa maintain pre-tRNA in an unprocessed state by blocking the 5'-processing site, impeding an early step in the pathway. Faithful phosphorylation of hLa on serine 366 reverses this block and promotes tRNA maturation. The results suggest that regulation of tRNA maturation at the level of RNase P cleavage may occur via phosphorylation of serine 366 of hLa.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: pre-steady-state kinetic studies

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine. The mechanism of the enzyme-catalyzed reaction was studied by isotope trapping, pre-steady-state rapid quench, and single turnover experiments. Isotope trapping indicated that the enzyme.tRNA complex is catalytically competent, whereas the enzyme.DMAPP complex is not. The results are consistent with an ordered sequential mechanism for substrate binding where tRNA binds first. The association and dissociation rate constants for the enzyme.tRNA binary complex are 1. 15+/-0.33x10(7) M(-1) s(-1) and 0.06+/-0.01 s(-1), respectively. Addition of DMAPP gives an enzyme.tRNA.DMAPP ternary complex in rapid equilibrium with the binary complex and DMAPP. Rapid quench studies yielded a linear profile (k(cat)=0.36+/-0.01 s(-1)) with no evidence for buildup of enzyme-bound product. Product release from DMAPP-tRNA transferase is therefore not rate-limiting. The Michaelis constant for tRNA and the equilibrium dissociation constant for DMAPP calculated from the individual rate constants determined here are consistent with values obtained from a steady-state kinetic analysis.     

A single tRNA (guanine)-methyltransferase from Tetrahymena with both mono- and di-methylating activity.

A tRNA (guanine-2) methyltransferase has been purified to homogeneity from the protozoan Tetrahymena pyriformis. The enzyme methylates purified E. coli tRNAs which have a guanine residue at position 26 from the 5' end; it also methylates tRNA prepared from the m22G- yeast mutant trm 1. This methyltransferase is therefore equivalent to the guanine methyltransferase 2mGII found in mammalian extracts. The purified 2mGII from Tetrahymena is capable of forming both N2-methylguanine and N22-dimethylguanine on a single tRNA isoaccepting species; under conditions of limiting tRNA or long reaction times the predominant product is dimethylguanine. Analysis of the products formed under varying reaction conditions suggests that dimethylguanine formation is a two step process requiring dissociation of the enzyme-monomethylated tRNA intermediate.     

The characterization of the RNAs and aminoacyl-tRNA synthetases of the blue-green alga, Anacystis nidulans

The tRNA and aminoacyl-tRNA synthetases of the blue-green alga, Anacystis nidulans have been isolated and studied. The distribution of some algal tRNA species on BD-cellulose chromatography has been determined. One tRNA^Met species has been isolated in 80% purity by a single chromatography on a BD-cellulose column developed with a modified salt gradient. The number of different tRNA isoacceptors for Met, Ser, and Leu has been ascertained by RPC-5 chromatography. The recognition of algal tRNAs by the homologous algal synthetase preparation as well as the heterologous Escherichia coli preparation was studied by the aminoacylation tests. Since all of the isoaccepting species of the tRNAs tested behaved almost identically in presence of the two enzyme preparations, a conservation of the recognition site during the evolutionary divergence of bacteria and algae is strongly suggested.     

The characterization of phosphoseryl tRNA from lactating bovine mammary gland.

BD-cellulose and RPC-5 chromatography of tRNA isolated from lactating bovine mammary gland showed the presence of four seryl-tRNA isoacceptors. The species, tRNA IV Ser, with the strongest affinity for BD-cellulose (required ethanol in the elution buffer) could be phosphorylated in the presence of serine, [gamma-32 P]-ATP, seryl-tRNA synthetase and phosphotransferase activity from the same tissue. O-Phosphoserine was identified as the 32P-labelled product after mild alkaline hydrolysis of this aminoacylated tRNA. Pancreatic ribonuclease treatment of the aminoacylated tRNA yielded a labelled product which was identified as phosphoseryladenosine. These results indicated there is a specific phosphoseryl tRNA species in lactating bovine mammary gland. It appears that the formation of phosphoseryl-tRNA proceeds by enzymic phosphorylation of seryl-tRNA.     

Human Tyrosine tRNA Is Also Internally Cleavable by E. coli Ribonuclease P RNA Ribozyme in Vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA^Ala, tRNA^His, and tRNA_i ^Met) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool to detect destablized tRNA molecules from any species.     

Affinity electrophoresis for monitoring terminal phosphorylation and the presence of queuosine in RNA. Application of polyacrylamide containing a covalently bound boronic acid

An affinity electrophoretic method has been developed to study the state of terminal phosphorylation of RNAs and the presence of the hypermodified base Q in tRNA. It is based on the copolymerization of acryloylaminophenylboronic acid into standard polyacrylamide gels and the interaction of this derivative with free cis-diol groups present in the RNA. In the case of terminal phosphorylation, free ribose groups are present either as such, or may be introduced by enzymatic reactions specific for a particular phosphorylation pattern (e.g. using T4 RNA ligase or guanylyltransferase). Additionally, tRNA species containing the Q base may be resolved from Q-lacking tRNAs by boronate affinity electrophoresis. The introduction of a non-destructive, one-step electrophoretic procedure not only offers an alternative to classical analytical methods, but also provides a means of isolating such populations of RNAs for which other methods are unavailable or are less convenient.     

Spermine stimulates the threonyl-tRNA formation in rat liver

The effects of spermine have been studied on the aminoacylation reaction catalyzed by rat liver threonyl-tRNA synthetase. Spermine can not replace Mg2+ in this reaction. However, a stimulatory and synergistic effect was observed on the threonyl-tRNA formation, in the presence of spermine and suboptimal concentration of Mg2+. Other divalent cations like Ba2+, Ca2+, Mn2+ and Co2+ can substitute Mg2+ in the threonyl-tRNA formation, but in all these cases spermine had no significant effect. Spermine prevented the inhibitory effects caused by excess of ATP or tRNA on the aminoacylation reaction. Association constants were determined by equilibrium dialysis for the tRNA-spermine complex (Ka = 3.7 x 10(3) M-1) and by differential spectrophotometry for the ATP-spermine complex (Ka = 7.8 x 10(3) M-1). No enzyme-spermine complex could be detected by equilibrium dialysis. Some roles have been ascribed for the polyamine spermine in the stimulation of the threonyl-tRNA formation. ATP-spermine and tRNA-spermine can not function as substrates for the threonyl-tRNA synthetase, since Mg2+ is indispensable. The stimulatory effect by spermine is important considering the physiological concentration of Mg2+ in the tissues. Probably in vivo spermine would have a relevant role lowering the real Mg2+ concentration required in the aminoacylation reaction.     

The distance of manganese to phosphorus atoms of polynucleotides, and dynamics of binding

The structure of manganese - polynucleotide complexes is studied by phosphorous NMR. An average value for the phosphorus-manganese distance is derived from the longitudinal relaxation rate. It is larger than the distance for direct coordination and decreases as a funtion of temperature. An inner-sphere/outer sphere equilibrium is proposed. The outer-sphere dominates in the double-stranded polynucleotides, whereas the inner-sphere contribution is important in single-stranded species. The kinetic parameters of the model are derived from the transverse relaxation time. The similar properties of DNA fragments and tRNA argue strongly against entrapment of manganese in special sites of tRNA.