Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 2. Preparation and ribosome interaction of fluorescent Escherichia coli tRNAMetf

A fluorescent derivative of tRNAMetf from Escherichia coli has been prepared which contains 1,N6-etheno-adenosine (epsilon A) in the place of adenosine 73, the fourth residue from the 3' end. The labeled tRNA, tRNAMetf epsilon A73, is fully active with respect to aminoacylation, formylation and formylmethionyl transfer to puromycin. The preparation procedure entails the chemical removal of four nucleotides from the 3' end of tRNAMetf, ligation of the truncated molecule with epsilon A 3',5'-bisphosphate by use of T4 RNA ligase and repair of the C-C-A end with nucleotidyl transferase. The fluorescence of fMet-tRNAMetf epsilon A73 has been exploited for studying tRNA-ribosome complexes. Upon binding the tRNA into the ribosomal P site, the fluorophor experiences a change of its molecular environment as indicated by an increased fluorescence intensity. On the other hand, iodide quenching experiments indicate that, in the complex, the fluorophor is not shielded against solvent access. The results suggest that (a) adenosine 73 is not involved in direct contacts with the ribosome and (b) the stacking of the 3'-terminal A-C-C-A sequence is changed upon binding to the ribosome.     

Optimization of conditions for labeling the 3' OH end of tRNA using T4 RNA ligase

For several years most primary structure studies of ribonucleic acids have used the [32P] in vitro post-labeling techniques. We adapted our methods from the literature, and simplified them to make them accessible to any laboratory. These procedures are especially useful for preparation and purification of post labeling enzymes: T4 polynucleotide kinase, T4 RNA ligase and of gamma [32P] ATP. We developed a test tube method for 5' [32P] pCp preparation followed by tRNA labeling with T4 RNA ligase. The parameters for optimal labeling were determined. Labeling of 3.10(6) to 5.10(6) Cerenkov CPM per microgram tRNA are currently obtained.     

Fluorescent labelling of tRNA and oligodeoxynucleotides using T4 RNA ligase.

3'-O-(5'-phosphoryldeoxycytidyl) phosphorothioate and fluorescent 3'-O-(5'-phosphoryldeoxycytidyl) S-bimane phosphorothioate can be ligated to tRNA by T4 RNA ligase. They are also efficient donors for the enzymatic ligation to oligodeoxynucleotides bearing a 3'-cytidine terminus. Cytidine 3',5'-bisphosphate is also a substrate for the ligation reaction with DNA restriction fragments with a 3'-terminate cytidylic acid residue. Oligo- and polynucleotides with a 3'-phosphorothioate group react readily with electrophiles as exemplified by the reaction with monobromobimane.     

Analogs of adenosine 3',5'-cyclic phosphate. II. Synthesis and enzymatic activity of derivatives of 1,N6-ethenoadenosine 3',5'-cyclic phosphate

The reactions of chloroacetaldehyde with adenosine 3′,5′-cyclic phosphate, and with several analogs modified at C₈ of the purine ring or C₅, of the sugar, lead to the corresponding 1,N⁶-etheno derivatives^d. Similar reactions using other 2-bromoaldehydes or phenacyl bromide give 1,N⁶-ethenonucleotides substituted at the α- or β-positions of the etheno bridge respectively. The ability of these compounds to activate the protein kinases from rabbit muscle and calf brain has been evaluated over a wide range of concentrations. While no derivative proved to be more active than adenosine 3′,5′-cyclic phosphate itself using the enzyme from rabbit muscle, a wide spectrum of activities was found using that from calf brain.     

Reactions at the termini of tRNA with T4 RNA ligase.

T4 RNA ligase will catalyze the addition of nucleoside 3', 5'-bisphosphates onto the 3' terminus of tRNA resulting in tRNA molecule one nucleotide longer with a 3' terminal phosphate. Under appropriate conditions the reaction is quantitative and, if high specific radioactivity bisphosphates are used, it provides an efficient means for in vitro labeling of tRNA. Although the 3' terminal hydroxyl is a good acceptor, the 5' terminal phosphate in most tRNA's is not an effective donor in the RNA ligase reaction. This poor reactivity is due to the secondary structure of the 5' terminal nucleotide. If E. Coli tRNAf Met is used, the 5' phosphate is reactive and the major product with RNA ligase is the cyclic tRNA.     

Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison

Related domains containing the purine NTP-binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N-terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N-ligase-kinase-phosphohydrolase-C, whereas polynucleotide kinase and CNPase encompass only the two C-terminal domains in the same order.     

The C-terminal domain of T4 RNA ligase 1 confers specificity for tRNA repair

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that thwarts a tRNA-damaging host response to virus infection. The 374-aa Rnl1 protein consists of an N-terminal nucleotidyltransferase domain fused to a unique C-terminal domain composed of 10 alpha helices. We exploited an in vitro tRNA splicing system to demonstrate that Rnl1 has an inherent specificity for sealing tRNA with a break in the anticodon loop. The tRNA specificity is imparted by the C domain, any deletion of which caused the broken tRNA to be sealed as poorly as the linear intron in vitro and also abolished Rnl1 tRNA splicing activity in vivo. Deletion analysis demarcated Rnl1-(1-254) as a minimal catalytic domain of Rnl1, capable of all chemical steps of the nonspecific RNA ligation reaction. Alanine scanning of the N domain identified Ser103, Leu104, Lys117, and Ser118 as important for pRNA ligation in vitro and tRNA repair in vivo.     

Incorporation of a kinin, N, 6-benzyladenine into soluble RNA

Kinin requiring tobacco and soybean tissues incubated on a medium containing N,6-benzyladenine-8-C¹⁴ incorporated C¹⁴ into several RNA components including adenylic and guanylic acids. About 15% of the label taken up by the tissues appeared in RNA while the remainder was distributed among several metabolites in the soluble, nonpolynucleotide fraction. Tissue grown on a kinin labeled in the side chain (N,6-benzyladenine-benzyl-C¹⁴) also incorporated a small, but nevertheless repeatable, amount of radioactivity into minor RNA components.

Ultracentrifugation studies and methylated albumin chromatography indicated that the bulk of the label from benzyladenine-benzyl-C¹⁴ is in soluble RNA. Approximately 50% of the C¹⁴ in soluble RNA is in a component which has chromatographic properties like that of benzyladenine.

It is suggested that the biological action of the kinins may hinge on their providing substituted bases in RNA in tissues which through differentiation no longer synthesize RNA-methylating enzymes. As an alternative it was hypothesized that a small amount of benzyladenine was incorporated into a m-RNA, acting there as a derepressing agent, perhaps by preventing its normal repressing function.

     

Biotin and fluorescent labeling of RNA using T4 RNA ligase.

Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules.     

Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA.

Host tRNAs cleaved near the anticodon occur specifically in T4-infected Escherichia coli prr strains which restrict polynucleotide kinase (pnk) or RNA ligase (rli) phage mutants. The cleavage products are transient with wt but accumulate in pnk- or rli- infections, implicating the affected enzymes in repair of the damaged tRNAs. Their roles in the pathway were elucidated by comparing the mutant infection intermediates with intact tRNA counterparts before or late in wt infection. Thus, the T4-induced anticodon nuclease cleaves lysine tRNA 5' to the wobble position, yielding 2':3'-P greater than and 5'-OH termini. Polynucleotide kinase converts them into a 3'-OH and 5' P pair joined in turn by RNA ligase. Presumably, lysine tRNA depletion, in the absence of polynucleotide kinase and RNA ligase mediated repair, underlies prr restriction. However, the nuclease, kinase and ligase may benefit T4 directly, by adapting levels or decoding specificities of host tRNAs to T4 codon usage.     

Protonation and quaternization of 1, N6-ethenoadenosine: What are the species responsible for the fluorescence of 1, N6-ethenoadenosine?

Methylation of 1,N⁶-ethenoadenosine (εAdo) gives a mixture of N1- and N9-quaternized methyl-3-β-D -ribofuranosylimidazo[2,1-i] purinium salts (m¹εAdo⁺ and m⁹εAdo⁺, respectively). The ratio of the two forms of the protonated εAdo [H¹εAdo⁺]/[H⁹εAdo⁺] has been estimated to be approximately 0.10 by comparing the uv absorption spectra of the protonated species of εAdo and the two nontautomerizable model compounds. In relation to a study on the protonation effect on the fluorescence of εAdo, we have now determined the effect of quaternization on the fluorescence spectra at 293 and 77 K. We have found that m¹εAdo⁺ and m⁹εAdo⁺ are both fluorescent, and the high degree of coincidence between the fluorescence spectra of εAdo and m¹εAdo⁺ at pH 7 is noted. The m¹εAdo⁺ singlet form is a more efficient fluorescer than the m⁹εAdo⁺ ion at room temperature (quantum yields of 0.43 and 0.11, respectively). All the results which are presented in this paper are consistent with the picture that there exist more than one species responsible for the fluorescence of εAdo, depending on the environment of the molecule in aqueous solution (temperature and pH of solvent).     

Reactions at the 3' terminus of transfer ribonucleic acid. IX. tRNA nucleotidyltransferase activity in bacteriophage T4-infected Escherichia coli

There was no detectable increase in tRNA nucleotidyltransferase activity upon infection of $\text{Escherichia coli}$ A19 with bacteriophage T4. Three mutant strains which contained low levels of tRNA nucleotidyltransferase activity also showed no increase in activity after infection. tRNA nucleotidyltransferase was purified from both uninfected and T4-infected cells and examined for possible modification. It was found that enzyme purified from both types of cells eluted from DEAE cellulose at the same specific conductivity. In addition, the molecular weight of tRNA nucleotidyltransferase purified from both uninfected and T4-infected cells was approximately 45,000 daltons as determined by chromatography on Sephadex G-100. These results suggest that T4-infection does not lead to synthesis of a new virus-specific tRNA nucleotidyltransferase nor does it cause modification of the host enzyme.     

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH(2)p) (100), clodronate (pCCl(2)p) (52), and etidronate (pC(OH)(CH(3))p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH(2)-CH(2)-NH(2))p), and alendronate (pC(OH)(CH(2)-CH(2)-CH(2)-NH(2))p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K(m) values and the rate of synthesis (k(cat)) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26+/-0.05 mM (0.28+/-0.05 s(-1)); clodronate, 0.54+/-0.14 mM (0.29+/-0.05 s(-1)); and etidronate, 4.3+/-0.5 mM (0.028+/-0.013 s(-1)). In the presence of GTP, and ATP or AppCCl(2)p the relative rate of synthesis of adenosine 5',5'-P(1),P(4)-tetraphosphoguanosine (Ap(4)G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH(2)p) was a very poor substrate for the synthesis of Ap(4)G. To our knowledge this report describes, for the first time, the synthesis of ATP beta,gamma-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.     

Di(1,N6-ethenoadenosine)5', 5'-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N6-ethenoadenosine)5',5'-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.     

Structure-guided mutational analysis of T4 RNA ligase 1

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.     

Evaluation of the intramolecular stacking of the fluorosulfonylbenzoyl derivatives of 1,N6-ethenoadenosine, adenosine, and guanosine

The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (? = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (? = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding protons of their parent nucleosides. The calculated difference in chemical shift (delta delta) decreases as the dielectric constant of the solvent decreases. The delta delta decreases with increasing temperature. These data indicate that 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG exist in aqueous solution in a conformation in which the purine ring is intramolecularly stacked with the benzoyl moiety. From the magnitude of change in delta delta for 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG as a function of solvent, it appears that the three analogues differ in their sensitivity to disruption of stacking. The solution conformation of these three fluorosulfonylbenzoyl nucleoside analogues may be an important determinant of their reaction with various enzymes and may explain differences among the analogues in their reaction with a single enzyme.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.     

Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase.

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.     

Investigations into the efficacy of 1,N6-ethenoadenosine triphosphate as a substrate for contractility studies

1,N⁶-Ethenoadenosine triphosphate was found to support superprecipitation of actomyosin and contraction of fibers. Its reactivity toward myosin parallels that of ITP. The relevance of these results in elucidating the mechanism for myosin nucleotide triphosphatase is discussed.