High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein.

Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain.     

Ribosomal RNA identity elements for ricin A-chain recognition and catalysis. Analysis with tetraloop mutants.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose of the adenosine at position 4324 in eukaryotic 28 S rRNA. Ricin A-chain will also catalyze depurination in naked prokaryotic 16 S rRNA; the adenosine is at position 1014 in a GAGA tetraloop. The rRNA identity elements for recognition by ricin A-chain and for the catalysis of cleavage were examined using synthetic GAGA tetraloop oligoribonucleotides. The RNA designated wild-type, an oligoribonucleotide (19-mer) that approximates the structure of the ricin-sensitive site in 16 S rRNA, and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type tetraloop oligoribonucleotide the ricin A-chain-catalyzed reaction has a Km of 5.7 microM and a Kcat of 0.01 min-1. The toxin alpha-sarcin, which cleaves the phosphodiester bond on the 3' side of G4325 in 28 S rRNA, does not recognize the tetraloop RNA, although alpha-sarcin does affect a larger synthetic oligoribonucleotide that has a 17-nucleotide loop with a GAGA sequence; thus, there is a clear divergence in the identity elements for the two toxins. Mutants were constructed with all of the possible transitions and transversions of each nucleotide in the GAGA tetraloop; none was recognized by ricin A-chain. Thus, there is an absolute requirement for the integrity of the GAGA sequence in the tetraloop. The helical stem of the tetraloop oligoribonucleotide can be reduced to three base-pairs, indeed, to two base-pairs if the temperature is decreased, without affecting recognition; the nature of these base-pairs does not influence recognition or catalysis by ricin A-chain. If the tetraloop is opened so as to form a GAGA-containing hexaloop, recognition by ricin A-chain is lost. This suggests that during the elongation cycle, a GAGA tetraloop either exists or is formed in the putative 17-member single-stranded region of the ricin domain in 28 S rRNA and this bears on the mechanism of protein synthesis.     

Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition.

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.     

Ricin A-chain substrate specificity in RNA, DNA, and hybrid stem-loop structures

Ricin toxin A-chain (RTA) is the catalytic subunit of ricin, a heterodimeric toxin from castor beans. Its ribosomal inactivating activity arises from depurination of a single adenine from position A(4324) in a GAGA tetraloop from 28S ribosomal RNA. Minimal substrate requirements are the GAGA tetraloop and stem of two or more base pairs. Depurination activity also occurs on stem-loop DNA with the same sequence, but with the k(cat) reduced 200-fold. Systematic variation of RNA 5'-G(1)C(2)G(3)C(4)[G(5)A(6)G(7)A(8)]G(9)C(10)G(11)C(12)-3' 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in k(cat) for A(6) --> dA(6) but reduced k(cat) up to 300-fold for the other sites. Methylation of individual 2'-hydroxyls in a similar experiment reduced k(cat) by as much as 3 x 10(-3)-fold. In stem-loop DNA, replacement of d[G(5)A(6)G(7)A(8)] with individual ribonucleotides resulted in small kinetic changes, except for the dA(6) --> A(6) replacement for which k(cat) decreased 6-fold. Insertion of d[G(5)A(6)G(7)A(8)] into an RNA stem-loop or G(5)A(6)G(7)A(8) into a DNA stem-loop reduced k(cat) by 30- and 5-fold, respectively. Multiple substitutions of deoxyribonucleotides into RNA stem-loops in one case (dG(5),dG(7)) decreased k(cat)/K(m) by 10(5)-fold, while a second change (dG(5),dA(8)) decreased k(cat) by 100-fold. Mapping these interactions on the structure of GAGA stem-loop RNA suggests that all the loop 2'-hydroxyl groups play a significant role in the action of ricin A-chain. Improved binding of RNA-DNA stem-loop hybrids provides a scaffold for inhibitor design. Replacing the adenosine of the RTA depurination site with deoxyadenosine in a small RNA stem-loop increased k(cat) 20-fold to 1660 min(-1), a value similar to RTA's k(cat) on intact ribosomes.     

Ribosomal RNA identity elements for ricin A-chain recognition and catalysis

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose at position A4324 in eukaryotic 28 S rRNA. The requirements for the recognition by ricin A-chain of this nucleotide and for the catalysis of cleavage were examined using a synthetic oligoribonucleotide that reproduces the sequence and the secondary structure of the RNA domain (a helical stem, a bulged nucleotide, and a 17-member single-stranded loop). The wild-type RNA (35mer) and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type oligoribonucleotide the ricin A-chain catalyzed reaction has a Km of 13.55 microM and a Kcat of 0.023 min-1. Recognition and catalysis by ricin A-chain has an absolute requirement for A at the position that corresponds to 4324. The helical stem is also essential; however, the number of base-pairs can be reduced from the seven found in 28 S rRNA to three without loss of identity. The nature of these base-pairs can affect catalysis. A change of the second set from one canonical (G.C) to another (U.A) reduces sensitivity to ricin A-chain; whereas, a change of the third pair (U.A----G.C) produces supersensitivity. The bulged nucleotide does not contribute to identification. Hydrolysis is affected by altering the nucleotides in the universal sequence surrounding A4324 or by changing the position in the loop of the tetranucleotide GA(ricin)GA: all of these mutants have a null phenotype. If ribosomes are treated first with alpha-sarcin to cleave the phosphodiester bond at G4325 ricin can still catalyze depurination at A4324. This implies that cleavage by alpha-sarcin at the center of what has been presumed to be a 17 nucleotide single-stranded loop in 28 S rRNA produces ends that are constrained in some way. On the other hand, hydrolysis by alpha-sarcin of the corresponding position in the synthetic oligoribonucleotide prevents recognition by ricin A-chain. The results suggest that the loop has a complex structure, affected by ribosomal proteins, and this bears on the function in protein synthesis of the alpha-sarcin/ricin rRNA domain.     

The ribosomal RNA identity elements for ricin and for alpha-sarcin: mutations in the putative CG pair that closes a GAGA tetraloop.

alpha-Sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3' side of G4325 in 28S rRNA; ricin A-chain is a RNA N-glycosidase that depurinates the 5' adjacent A4324. These single covalent modifications inactivate the ribosome. An oligoribonucleotide that reproduces the structure of the sarcin/ricin domain in 28S rRNA was synthesized and mutations were constructed in the 5' C and the 3' G that surround a GAGA tetrad that has the sites of toxin action. Covalent modification of the RNA by ricin, but not by alpha-sarcin, requires a Watson-Crick pair to shut off a putative GAGA tetraloop. Either the recognition elements for the two toxins are different despite their catalyzing covalent modification of adjacent nucleotides in 28S rRNA or there are transitions in the conformation of the alpha-sarcin/ricin domain in 28S rRNA and one conformer is recognized by alpha-sarcin and the other by ricin A-chain.     

The RNA N-glycosidase activity of ricin A-chain

The RNA N-glycosidase activity of ricin A-chain has been characterized. When rat liver ribosomes were used as substrates, the A-chain cleaved the N-glycosidic bond at A-4324 in 28S rRNA. An apparent Michaelis constant (Km) for the reaction was determined to be 2.6 microM and the turnover number (Kcat) was 1777 min-1. When naked rRNA was the substrate, the A-chain cleaved the same bond in 28S rRNA but at a greatly reduced rate. The Km value was 5.8 microM. The results suggest that the A-chain has a similar affinity for 28S rRNA in both ribosomes and the naked states. When the deproteinized Escherichia coli rRNA was the substrates, ricin A-chain cleaved a N-glycosidic bond at A-2600 in 23S rRNA which corresponds to the ricin-site in 28S rRNA of rat liver ribosomes, while the A-chain has little activity on 23S rRNA in the ribosomes. The results suggest that ricin A-chain acts directly on RNA by recognizing a certain structure in the molecules. Using the secondary structure models for each species of rRNA, we have deduced a loop and stem structure having GAGA in the loop to be a minimum requirement for the substrate of ricin A-chain.     

Ricin A-chain activity on stem-loop and unstructured DNA substrates

Ricin toxin A-chain (RTA) depurinates a single adenylate on a GAGA stem-loop region of eukaryotic 28S RNA, making it a potent toxin. Steady state rate analysis is used to establish the kinetic parameters for depurination of short RNA, DNA, and RNA-DNA hybrids of GAGA linear segments and stem-loop regions as substrates for RTA. Both stem and tetraloop structures are essential for action on RNA. For DNA stem-loop substrates, stem stability plays a small role in enhancing catalytic turnover but can enhance binding by up to 3 orders of magnitude. DNA sequences of d[GAGA] without stem-loop structures are found to be slow substrates for RTA. In contrast, equivalent RNA sequences exhibit no activity with RTA. Introduction of a deoxyadenosine at the depurination site of short RNA oligonucleotides restores catalytic function. NMR analysis indicates that the short, nonsubstrate GAGA is converted to substrate in GdAGA by the presence of a more flexible ribosyl group at the deoxyadenosine site. Conversion between C2'-endo and C2'-exo conformations at the deoxyadenosine site moves the 3'- and 5'-phosphorus atoms by 1.1 A, and the former is proposed to place them in a catalytically favorable configuration. The ability to use short RNA-DNA hybrids as substrates for RTA permits exploration of related structures to function as substrates and inhibitors.     

The consensus sequence for self-catalyzed site-specific G residue depurination in DNA

The sequence variation tolerated within the stem-loop-forming genomic consensus sequence for self-catalyzed site-specific depurination of G residues is explored. The variation in self-depurination kinetics with sequence changes in the loop residues and stem base pairs, as well as with pH, provides insights into the self-catalytic mechanism. The observations suggest that self-catalyzed depurination of the 5' G residue of the loop consensus sequence 5'-G(T/A)GG-3' probably involves formation of some intraloop hydrogen-bonded base pair with the 3'-terminal G residue; although the electronic structure of both these G residues is retained, their 2-amino substituents are not critical for that interaction. The strong dependence of the self-depurination kinetics on stem stability suggests that the lifetime of some strained form of the loop is controlled by the integrity of the stem. In addition to the effects of length and base pair sequence on stem stability, there is a base pair requirement at the base of the loop: self-depurination is suppressed by 5'-C·G-3', 5'-A·T-3', or a mismatch but is most favored by 5'T·A3' and less so by 5'-G·C-3'. The occurrence in T and G of a similarly located carbonyl capable of hydrogen-bonding to the water molecule required for glycosyl bond hydrolysis may explain this sequence requirement. In toto, the more complete definition of the consensus sequence provided by this investigation enables a more accurate estimation of their number in the human genome and their distribution among different genes.     

Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain

8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.