ABSTRACT
Small ribozymes use their nucleobases to catalyse phosphodiester bond cleavage. The hepatitis delta virus ribozyme employs C75 as a general acid to protonate the 5'-bridging oxygen leaving group, and to accomplish this task efficiently, it shifts its pKa towards neutrality. Simulations and thermodynamic experiments implicate linkage between folding and protonation in nucleobase pKa shifting. Even small oligonucleotides are shown to fold in a highly co-operative manner, although they do so in a context-specific fashion. Linkage between protonation and co-operativity of folding may drive pKa shifting and provide for enhanced function in RNA.
Subject(s)
Nucleic Acid Conformation , Protons , RNA/chemistry , RNA/metabolism , Base Sequence , Catalysis , Hepatitis Delta Virus/enzymology , Hepatitis Delta Virus/genetics , RNA, Catalytic/chemistry , RNA, Catalytic/metabolismABSTRACT
Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HDV contain a ribozyme that undergoes cis-cleavage, thereby processing the transcript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have attenuated self-cleavage. Structure mapping and site-directed mutagenesis revealed an inhibitory stretch consisting of upstream nucleotides -24 to -15 that forms a long-range pairing, termed Alt 1, with the 3' strand of P2 (P2(3')) located at the very 3'-end of the ribozyme. Two other alternative pairings were found, Alt 2, which involves upstream nucleotide-ribozyme interactions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleavage was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the -24/-15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased to 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides -54 to -18, termed P(-1)/L(-1), which sequesters the majority of the -24/-15 inhibitory stretch in cis. This hairpin is followed by a stretch of single-stranded pyrimidine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, and that the J(-1/1) stretch is conserved among antigenomic HDV isolates. Lastly, the secondary structure of the Alt 1-containing ribozyme provides insight into possible folding intermediates of the ribozyme.
Subject(s)
Hepatitis Delta Virus/chemistry , RNA, Catalytic/chemistry , RNA, Viral/chemistry , Algorithms , Amino Acid Motifs , Base Sequence , Conserved Sequence , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid ConformationABSTRACT
Many protein enzymes use general acid-base catalysis as a way to increase reaction rates. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH. The RNA enzyme (ribozyme) from hepatitis delta virus catalyzes self-cleavage of a phosphodiester bond. Reactivity-pH profiles in monovalent or divalent cations, as well as distance to the leaving-group oxygen, implicate cytosine 75 (C75) of the ribozyme as the general acid and ribozyme-bound hydrated metal hydroxide as the general base in the self-cleavage reaction. Moreover, C75 has a pK(a) perturbed to neutrality, making it "histidine-like." Anticooperative interaction is observed between protonated C75 and a metal ion, which serves to modulate the pK(a) of C75. General acid-base catalysis expands the catalytic repertoire of RNA and may provide improved rate acceleration.
Subject(s)
Hepatitis Delta Virus/chemistry , RNA, Catalytic/metabolism , Base Pairing , Binding Sites , Calcium/metabolism , Catalysis , Cobalt/metabolism , Crystallography, X-Ray , Hepatitis Delta Virus/enzymology , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Magnesium/metabolism , Metals/metabolism , Models, Chemical , Models, Molecular , Nucleic Acid Conformation , Protons , RNA, Catalytic/chemistry , RNA, Viral/chemistry , RNA, Viral/metabolism , Static Electricity , ThermodynamicsABSTRACT
An acid alpha-glucosidase (EC 3.2.1.3) has been purified to electrophoretic homogeneity from the soluble fraction of the human term placenta. In the presence of SDS, two doublets of 79 and 67 kDa were seen in addition to other bands of extremely low intensity. Each of these bands was seen to cross-react with polyclonal antiserum raised to the purified enzyme, thus confirming the homogeneity of the preparation. The purified enzyme exhibited a broad substrate specificity. The kinetic data revealed the possible presence of multiple substrate binding sites. Chemical modification using group specific reagents indicated the presence of a carboxyl group and tryptophan at the active site. Based on these results a possible structure for the active site of the human term placental acid alpha-glucosidase has been proposed.
