Martha J. Fedor

The catalytic diversity of RNAs

The natural RNA enzymes catalyse phosphate-group transfer and peptide-bond formation. Initially, metal ions were proposed to supply the chemical versatility that nucleotides lack. In the ensuing decades, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly rely on essential metal-ion cofactors, self-cleaving ribozymes seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences in chemical features, both RNA and protein enzymes use similar catalytic strategies.

Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast

Arrays of nucleosomes were positioned with respect to the GAL1-GAL10 intergenic region inserted into Saccharomyces cerevisiae minichromosomes. Deletions of DNA flanking the upstream activation sequence left the array unaltered, showing that nucleosome positioning was not a consequence of sequence-specific histone-DNA interactions but depended on proximity to the galactose-responsive upstream activation sequence (UASG). Replacement of the upstream activation sequence by synthetic oligonucleotides with different protein-binding properties identified a short sequence within this region that is responsible for the ordered array. This sequence overlaps a binding site for GAL4 protein, a positive regulator of transcription, but exerts its effect on chromatin structure independently of GAL4, probably through binding a novel factor that is not GAL-specific.

Comparative Enzymology and Structural Biology of RNA Self-Cleavage

Self-cleaving hammerhead, hairpin, hepatitis delta virus, and glmS ribozymes comprise a family of small catalytic RNA motifs that catalyze the same reversible phosphodiester cleavage reaction, but each motif adopts a unique structure and displays a unique array of biochemical properties. Recent structural, biochemical, and biophysical studies of these self-cleaving RNAs have begun to reveal how active site nucleotides exploit general acid-base catalysis, electrostatic stabilization, substrate destabilization, and positioning and orientation to reduce the free energy barrier to catalysis. Insights into the variety of catalytic strategies available to these model RNA enzymes are likely to have important implications for understanding more complex RNA-catalyzed reactions fundamental to RNA processing and protein synthesis.

The role of metal ions in RNA catalysis

Understanding the catalytic mechanisms of RNA enzymes remains an important and intriguing challenge - one that has grown in importance since the recent demonstration that the ribosome is a ribozyme. At first, it seemed that all RNA enzymes compensate for the limited chemical versatility of ribonucleotide functional groups by recruiting obligatory metal ion cofactors to carry out catalytic chemistry. Mechanistic studies of the large self-splicing and pre-tRNA-processing ribozymes continue to support this idea, yielding increasingly detailed views of RNA active sites as scaffolds for positioning catalytic metal ions. Re-evaluation of the methodologies used to distinguish catalytic and structural roles for metal ions, however, has challenged this notion in the case of the small self-cleaving RNAs. Recent studies of the small ribozymes blur the distinction between catalytic and structural roles for metal ions, and suggest that RNA nucleobases have a previously unrecognized capacity for mediating catalytic chemistry.

mRNA Secondary Structures Fold Sequentially But Exchange Rapidly In Vivo

Self-cleavage assays of RNA folding reveal that mRNA structures fold sequentially in vitro and in vivo, but exchange between adjacent structures is much faster in vivo than it is in vitro.

Upstream activation sequence-dependent alteration of chromatin structure and transcription activation of the yeast GAL1-GAL10 genes.

Conversion of the positioned nucleosome array characteristic of the repressed GAL1-GAL10 promoter region to the more accessible conformation of the induced state was found to depend on the upstream activation sequence, GAL4 protein, a positive regulator of transcription, and galactose, the inducing agent. The effect of the GAL4 protein-upstream activation sequence complex on the structure of adjacent chromatin required no other promoter sequences. Although sequences protected by histones in the repressed state became more accessible to micrococcal nuclease and (methidiumpropyl-EDTA)iron(II) cleavage following induction of transcription, DNA-protein particles containing these sequences retained the electrophoretic mobility of nucleosomes, indicating that the promoter region can be associated with nucleosomes under conditions of transcription activation.

Rescue of an Abasic Hairpin Ribozyme by Cationic Nucleobases: Evidence for a Novel Mechanism of RNA Catalysis

The hairpin ribozyme catalyzes a reversible phosphodiester cleavage reaction. We examined the roles of conserved nucleobases in catalysis using an abasic ribozyme rescue strategy. Loss of the active site G8 nucleobase reduced the cleavage rate constant by 350-fold while loss of A9 and A10 nucleobases reduced activity less than 10-fold. Certain heterocyclic amines restored partial activity when provided in solution to the variant lacking G8. Heterocyclic amines that were capable of rescue shared the exocyclic amine and cyclic amide in common with the Watson-Crick hydrogen bonding face of guanine. In contrast to the shallow pH dependence of unmodified ribozyme activity, rescue activity increased sharply with decreasing pH. These results support a novel model for RNA catalysis in which a cationic nucleobase contributes electrostatic stabilization to negative charge developing in the transition state.

The glmS riboswitch integrates signals from activating and inhibitory metabolites in vivo

The glmS riboswitch belongs to the family of regulatory RNAs that provide feedback regulation of metabolic genes. It is also a ribozyme that self-cleaves upon binding glucosamine-6-phosphate, the product of the enzyme encoded by glmS. The ligand concentration dependence of intracellular self-cleavage kinetics was measured for the first time in a yeast model system and unexpectedly revealed that this riboswitch is subject to inhibition as well as activation by hexose metabolites. Reporter gene experiments in Bacillus subtilis confirmed that this riboswitch integrates positive and negative chemical signals in its natural biological context. Contrary to the conventional view that a riboswitch responds to just a single cognate metabolite, our new model proposes that a single riboswitch integrates information from an array of chemical signals to modulate gene expression based on the overall metabolic state of the cell.

Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise function(s) in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8 and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.

Adenovirus chromatin structure at different stages of infection

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.