The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.     

In vitro selection of dopamine RNA ligands

RNA aptamers that specifically bind dopamine have been isolated by in vitro selection from a pool of 3.4 x 10(14) different RNA molecules. One aptamer (dopa2), which dominated the selected pool, has been characterized and binds to the dopamine affinity column with a dissociation constant of 2.8 microM. The specificity of binding has been determined by studying binding properties of a number of dopamine-related molecules, showing that the interaction with the RNA might be mediated by the hydroxyl group at position 3 and the proximal aliphatic chain in the dopamine molecule. The binding domain was initially localized by boundary experiments. Further definition of the dopamine binding site was obtained by secondary selection on a pool of sequences derived from a partial randomization of the dopa2 molecule. Sequence comparison of a large panel of selected variants revealed a structural consensus motif among the active aptamers. The dopamine binding pocket is built up by a tertiary interaction between two stem and loop motifs, creating a stable framework in which five invariant nucleotides are precisely arrayed. Minimal active sequence and key nucleotides have been confirmed by the design of small functional aptamers and mutational analysis. Enzymatic probing suggests that the RNA might undergo a conformational change upon ligand binding that stabilizes the proposed tertiary structure.     

Structure-function analysis in nuclear RNase P RNA

Eukaryotic ribonuclease P (RNase P) enzymes require both RNA and protein subunits for activity in vivo and in vitro. We have undertaken an analysis of the complex RNA subunit of the nuclear holoenzyme in an effort to understand its structure and its similarities to and differences from the bacterial ribozymes. Phylogenetic analysis, structure-sensitive RNA footprinting, and directed mutagenesis reveal conserved secondary and tertiary structures with both strong similarities to the bacterial consensus and distinctive features. The effects of mutations in the most highly conserved positions are being used to dissect the functions of individual subdomains.     

Mitochondrial RNase P: the RNA family grows

Recent results of biochemical approaches and genome sequencing approaches has extended the members of the family of mitochondrial RNase P RNA genes. So far all of them are AU rich, and most of their secondary structures are easier to predict than was the structure of the first mitochondrial RNase P RNA from S. cerevisiae. The recently sequenced protozoan R. americana mitochondrial gene displays many of the evolutionarily conserved primary sequence and secondary structure attributes of the well characterized bacterial RNase P RNAs. Continued addition of RNAs to this data base should allow increasingly informative alignments and an understanding of what structural elements are dispensable in the smallest mitochondrial RNAs. The only protein subunits identified to date are Rpm2p from S.cerevisiae and the homologous protein from S. douglasii.     

An active domain of the nuclear RNase P RNA

The P10/11-P12 RNA domain of yeast nuclear RNase P RNA has been characterized using genetic and biochemical analysis. This RNA domain contains some of the most conserved nucleotides throughout yeast species and shares considerable homology with the P10-P11-P12 bacterial RNase P RNA domain. Viable yeast variants generated by sequence randomization of the conserved internal loop nucleotides have demonstrated magnesium-sensitive growth defects. Partial purification and characterization of the RNase P holoenzyme from these variants reveals that the mutations affect the catalytic rate of the enzyme and increased magnesium concentrations are required to achieve maximal activity compared to wild type enzyme. Biochemical structure probing has been employed to address the interaction of the RNA domain with magnesium. Several nucleotides within the loop portion of the domain show magnesium-induced changes in reagent accessibility. These include the highly conserved nucleotides shared between yeast and bacteria, which become less accessible in the presence of magnesium. Conversely, accessibility of other regions of the RNA increases. The genetic and biochemical data suggest that the P10/11-P12 RNA domain, and the conserved nucleotides in particular, interacts with magnesium in a manner that affects catalysis by RNase P.     

In vitro selection of bacteriophage phi29 prohead RNA aptamers for prohead binding

Prohead RNA (pRNA) of the Bacillus subtilis bacteriophage phi29 is needed for in vitro packaging of DNA-gene product 3 (DNA-gp3). Residues 22-84 of the 174-base pRNA bind the portal vertex of the prohead, the site of DNA packaging. To define the nucleotides of pRNA needed for prohead binding and DNA-gp3 packaging and to seek biologically active variants of pRNA, segments of pRNA were randomized to obtain vast repertoires of RNA molecules. RNA aptamers, ligands best suited for prohead binding, were obtained by multiple rounds of in vitro selection. Evolution of pRNA aptamers was followed by a competition binding assay and nucleotide sequencing, and mutants were tested for DNA-gp3 packaging. Aptamers selected following randomization of the E stem and loop and a part of the C-E loop that were active in DNA-gp3 packaging were invariably wild-type. DNA-gp3 packaging activity also required nucleotides G82 and G83 that form base pairs intermolecularly with C47 and C48 to produce a novel hexameric oligomer of pRNA. The only mutant aptamers that retained full DNA-gp3 packaging activity showed changes of the U residues at positions 81, 84, and 85 of the D loop. Thus, the in vitro selections essentially recapitulated the natural evolution of pRNA.     

Nucleotide and divalent cation specificity of in vitro iron-molybdenum cofactor synthesis

The nucleotide and divalent cation requirements of the in vitro iron-molybdenum cofactor (FeMo-co) synthesis system have been compared with those of substrate reduction by nitrogenase. The FeMo-co synthesis system specifically requires ATP, whereas both 1,N6-etheno-ATP and 2'-deoxy-ATP function in place of ATP in substrate reduction (M. F. Weston, S. Kotake, and L. C. Davis, Arch. Biochem. Biophys. 225:809-817, 1983). Mn2+, Ca2+, and Fe2+ substitute for Mg2+ to various extents in in vitro FeMo-co synthesis, whereas Ca2+ is ineffective in substrate reduction by nitrogenase. The observed differences in the nucleotide and divalent cation specificities suggest a role(s) for the nucleotide and divalent cation in in vitro FeMo-co synthesis that is distinct from their role(s) in substrate reduction.     

Enzymatic RNA synthesis and RNase P. Evolutionary aspects

TransferRNA recognition was used as leit-motiv in the illustration of possible links between a hypothetical primordial RNA world and the contemporary DNA world. In an RNA world, 'proto-tRNA' could have functioned as replication origin and as primitive telomere. Possibly, this primitive structure is preserved in a 'universal substrate' for modern tRNA-specific enzymes. The combination of acceptor stem and T arm (plus a linker) was finally revealed as sufficient for the recognition by prokaryotic and eukaryotic RNase P, as well as other tRNA enzymes. In modern life forms, a tRNA-like element in viral RNAs still serves as replication origin, and furthermore, the recognition of similar structures as cryptic promoters is universally conserved for template-dependent RNA polymerases. Another common property of modern polymerases is their high, but clearly limited and condition-dependent substrate specificity. Very likely, also substrate recognition by primitive polymerases was not more stringent, and this lead to the occurrence of mixed nucleic acids as intermediates in the transition of genomic RNA to contemporary genomic DNA.     

In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.     

In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution

BACKGROUND: It has been hypothesized that the fact that both ribosomal RNA and the group I intron can bind to aminoglycoside antibiotics implies that these RNAs are evolutionarily related. This hypothesis requires the assumption that there are relatively few ways for RNA molecules to form aminoglycoside-binding sites. RESULTS: We have used in vitro selection to determine the diversity of aminoglycoside-binding sites that can be formed by RNA molecules. We have generated RNA 'lectins' that can bind aminoglycosides tightly and specifically. Sequence analysis indicates that there are many different ways to form tight and specific aminoglycoside binding sites. These artificially selected binding sites are functionally similar to those that have arisen from natural selection. CONCLUSIONS: Our results suggest that the presence of aminoglycoside-binding sites on RNA molecules may not be a useful trait for determining evolutionary relatedness. Instead, the fact that RNA molecules can bind these 'low molecular-weight effectors' may indicate that natural products such as aminoglycosides have evolved to exploit sequence- and structure-specific recognition of nucleic acids, in much the same way that lexitropsins have been designed by chemists to recognise specific nucleic acid sequences.     

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.     

In vitro selection of phosphorothiolated aptamers

A pool of RNA molecules that contained exclusively phosphorothioate internucleoside linkages was used as a starting point for the selection of aptamers that bind to basic fibroblast growth factor (bFGF), and appear to act as heparin mimics.     

In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules

Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues. The methylated guanosine residue closely resembles the 5' terminal cap structure present on all eukaryotic mRNA molecules. The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from Saccharomyces cerevisiae. These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.     

Evolution of a phosphorothioate RNA library during in vitro selection

We carried out an in vitro selection of aptamers from an RNA library with phosphorothioate linkages instead of normal phosphodiesters. This "thio-RNA" library had smaller dissociation rate constant (kd) to a target protein than a natural RNA library. On the basis of the results, an improved selection procedure to obtain ideal nucleic acid drugs will be discussed.     

In vitro selection analysis of trans-acting HDV ribozyme

In order to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. After 10 generations, a randomized pool of trans-acting ribozymes accumulated in which the secondary structure of each ribozyme confirmed to the pseudoknot model and important bases in single-stranded regions were all conserved. We were surprised that mutated ribozymes derived from genomic sequence were changed to anti-genomic-like sequences. Further investigations of the most active variant confirmed that each mutated base was the most appropriate nucleotide at every position of HDV ribozyme.     

Evolutionary variation in bacterial RNase P RNAs

Sequences encoding RNase P RNAs from representatives of the last remaining classical phyla of Bacteria have been determined, completing a general phylogenetic survey of RNase P RNA sequence and structure. This broad sampling of RNase P RNAs allows some refinement of the secondary structure, and reveals patterns in the evolutionary variation of sequences and secondary structures. Although the sequences range from 100 to <25% identical to one another, and although only 40 of the nucleotides are invariant, there is considerable conservation of the underlying core of the RNA sequence. RNase P RNAs, like group I intron RNAs but unlike ribosomal RNAs, transfer RNAs or other highly conserved RNAs, are quite variable in secondary structure outside of this conserved structural core. Conservative regions of the RNA evolve by substitution of apparently interchangeable alternative structures, rather than the insertion and deletion of helical elements that occurs in the more variable regions of the RNA. In a remarkable case of convergent molecular evolution, most of the unusual structural elements of type B RNase P RNAs of the low G+C Gram-positive Bacteria have evolved independently in Thermomicrobium roseum , a member of the green non-sulfur Bacteria.     

Evidence for an RNA binding region in the Escherichia coli processing endoribonuclease RNase E

The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA. A number of deletions were constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides. The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants. Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region. Binding to substrate RNAs correlated with the endonucleolytic activity. RNAs that are not substrates for RNase E did not bind to the protein. The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished. The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity.