DNA and RNA topoisomerase activities of Top3ß are promoted by mediator protein Tudor domain-containing protein 3.

Topoisomerase 3ß (Top3ß) can associate with the mediator protein Tudor domain-containing protein 3 (TDRD3) to participate in two gene expression processes of transcription and translation. Despite the apparent importance of TDRD3 in binding with Top3ß and directing it to cellular compartments critical for gene expression, the biochemical mechanism of how TDRD3 can affect the functions of Top3ß is not known. We report here sensitive biochemical assays for the activities of Top3ß on DNA and RNA substrates in resolving topological entanglements and for the analysis of TDRD3 functions. TDRD3 stimulates the relaxation activity of Top3ß on hypernegatively supercoiled DNA and changes the reaction from a distributive to a processive mode. Both supercoil retention assays and binding measurement by fluorescence anisotropy reveal a heretofore unknown preference for binding single-stranded nucleic acids over duplex. Whereas TDRD3 has a structure-specific binding preference, it does not discriminate between DNA and RNA. This unique property for binding with nucleic acids can have an important function in serving as a hub to form nucleoprotein complexes on DNA and RNA. To gain insight into the roles of Top3ß on RNA metabolism, we designed an assay by annealing two single-stranded RNA circles with complementary sequences. Top3ß is capable of converting two such single-stranded RNA circles into a double-stranded RNA circle, and this strand-annealing activity is enhanced by TDRD3. These results demonstrate that TDRD3 can enhance the biochemical activities of Top3ß on both DNA and RNA substrates, in addition to its function of targeting Top3ß to critical sites in subcellular compartments.

The poly(A) site sequence in HDV RNA alters both extent and rate of self-cleavage of the antigenomic ribozyme.

The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1-2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.

Essential role of an active-site guanine in glmS ribozyme catalysis.

The glmS ribozyme is a catalytic riboswitch that is activated for endonucleolytic cleavage by the coenzyme glucosamine-6-phosphate. Using kinetic assays and X-ray crystallography, we identify an active-site mutation of a conserved guanine that abolishes catalysis without perturbing coenzyme binding. Our results provide evidence that coenzyme function requires a specific nucleobase to interact with the nucleophile of the cleavage reaction.

Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme.

The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

A single nucleotide linked to a switch in metal ion reactivity preference in the HDV ribozymes.

The two ribozymes of hepatitis delta virus (HDV) cleave faster in divalent metal ions than in monovalent cations, and a variety of divalent metal ions can act as catalysts in supporting these higher rates. Although the ribozymes are closely related in sequence and structure, they display a different metal ion preference; the genomic form cleaves moderately faster in Mg2+ than in Ca2+ while the reverse is true for the antigenomic ribozyme. This difference raises questions about understanding the catalytic role of the metal ion in the reaction. We found that the metal ion reactivity preference correlated with the identity of a single nucleotide 5' of the cleavage site (-1 position). It is a U in the genomic sequence and a C in the antigenomic sequence. With both ribozymes, the reactivity preference for Mg2+ and Ca2+ could be reversed with a change at this position (C to U or U to C). Moreover, with an A at position -1, there was a relative increase in cleavage rates in low concentrations of Mn2+ for both ribozymes. Metal ion reactivity preference was also linked to changes in pH, and the pH-rate profiles could be shifted with nucleotide changes at position -1. Together, the data provide biochemical evidence in support of an organized active site, as seen in the crystal structures, where at least one metal ion, an ionizable group, and the conformation of the phosphate backbone at the cleavage site interact in concert to promote cleavage.

HDV ribozyme activity in monovalent cations.

Activity of the two ribozymes from hepatitis delta virus in monovalent salts was examined and compared to activity in Mg2+. Both ribozymes self-cleaved in high concentrations of monovalent cations, and an active site cytosine was required for cleavage activity under those conditions. Cleavage rates were 30-50-fold higher for reactions in LiCl than for reactions in NaCl or NH4Cl, and a thio effect indicated that chemistry was rate-determining for cleavage of the HDV genomic ribozyme in LiCl. Still, in LiCl, there was a more than 100-fold increase in the rate when MgCl2 was included in the reaction. However, the pH-rate profiles for the reactions in LiCl with and without MgCl2 were both bell-shaped with the pH optima in the neutral range. These findings support the idea that monovalent cations can partially substitute for divalent metal ions in the HDV ribozymes, although a divalent metal ion is more effective in supporting catalysis. The absence of a dramatic change in the general shape of pH-rate profiles in LiCl, relative to the profile for reactions including Mg2+, is in contrast to earlier data for the reactions in NaCl and limits our interpretation of the specific role played by the divalent metal ion in the catalytic mechanism.

Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme.

In the ribozyme from the hepatitis delta virus (HDV) genomic strand RNA, a cytosine side chain is proposed to facilitate proton transfer in the transition state of the reaction and, thus, act as a general acid-base catalyst. Mutation of this active-site cytosine (C75) reduced RNA cleavage rates by as much as one million-fold, but addition of exogenous cytosine and certain nucleobase or imidazole analogs can partially rescue activity in these mutants. However, pH-rate profiles for the rescued reactions were bell shaped, and only one leg of the pH-rate curve could be attributed to ionization of the exogenous nucleobase or buffer. When a second potential ionizable nucleobase (C41) was removed, one leg of the bell-shaped curve was eliminated in the chemical-rescue reaction. With this construct, the apparent pK(a) determined from the pH-rate profile correlated with the solution pK(a) of the buffer, and the contribution of the buffer to the rate enhancement could be directly evaluated in a free-energy or Brønsted plot. The free-energy relationship between the acid dissociation constant of the buffer and the rate constant for cleavage (Brønsted value, beta, = approximately 0.5) was consistent with a mechanism in which the buffer acted as a general acid-base catalyst. These data support the hypothesis that cytosine 75, in the intact ribozyme, acts as a general acid-base catalyst.

A pseudoknot in the 3' non-core region of the glmS ribozyme enhances self-cleavage activity.

The recently described glmS ribozyme is a self-cleaving RNA sequence found in the 5' noncoding region of the transcript of the gene for glucosamine-6-phosphate (GlcN6P) synthase in many Gram-positive bacteria. This ribozyme is associated with the GlcN6P riboswitch, and ribozyme activity in response to binding of the metabolite, GlcN6P, is proposed to effect levels of gene expression. The previously defined core sequence of the GlcN6P-dependent ribozyme contained fewer than 80 nt of contiguous sequence, but a sequence containing conserved secondary structural features and encompassing the core was twice as long. Structural elements outside of the ribozyme core could contribute to ribozyme activity or participate in gene regulation as part of the expression platform or both. Here, a 174-nt transcript containing the Bacillus anthracis glmS ribozyme was used to examine the contribution of part of the non-core sequence to in vitro cleavage activity. The loop portion of hairpin loop 3, located just 3' of the ribozyme core, can potentially pair with a sequence approximately 80 nt downstream to form a pseudoknot tertiary interaction. Disruptive and compensatory mutations in the two duplex regions of the pseudoknot had effects on in vitro cleavage rates that support a role for the pseudoknot in enhanced ribozyme activity. Cleavage activity became less sensitive to disruptive mutations in the pseudoknot as MgCl(2) concentrations were raised from 2.5 to 10 mM, suggesting that one role of the pseudoknot could be to help stabilize the core structure.

Catalytic strategies of the hepatitis delta virus ribozymes.

The hepatitis delta virus (HDV) ribozymes are self-cleaving RNA sequences critical to the replication of a small RNA genome. A recently determined crystal structure together with biochemical and biophysical studies provides new insight into the possible catalytic mechanism of these ribozymes. The HDV ribozymes are examples of naturally occurring small ribozymes that catalyze cleavage of the RNA backbone with a rate enhancement of 10(6)- to 10(7)-fold over the uncatalyzed rate. To achieve this level of rate enhancement, the HDV ribozymes have been proposed to employ several catalytic strategies that include the use of metal ions, intrinsic binding energy, and a novel example of general acid-base catalysis with a cytosine side chain acting as a proton donor or acceptor.