The structure of ribonuclease P protein from Staphylococcus aureus reveals a unique binding site for single-stranded RNA.

Ribonuclease P (RNaseP) catalyses the removal of the 5'-leader sequence from pre-tRNA to produce the mature 5' terminus. The prokaryotic RNaseP holoenzyme consists of a catalytic RNA component and a protein subunit (RNaseP protein), which plays an auxiliary but essential role in vivo by binding to the 5'-leader sequence and broadening the substrate specificity of the ribozyme. We determined the three-dimensional high-resolution structure of the RNaseP protein from Staphylococcus aureus (117 amino acid residues) by nuclear magnetic resonance (NMR) spectroscopy in solution. The protein has an alphabeta-fold, similar to the ribonucleoprotein domain. We used small nucleic acid molecules as a model for the 5'-leader sequence to probe the propensity for generic single-stranded RNA binding on the protein surface. The NMR results reveal a contiguous interaction site, which is identical with the previously identified leader sequence binding site in RNaseP holoenzyme. The conserved arginine-rich motif does not bind single-stranded RNA. It is likely that this peptide segment binds selectively to double-stranded sections of P RNA, which are conformationally more rigid. Given the essentiality of RNaseP for the viability of the organism, knowledge of the S. aureus protein structure and insight into its interaction with RNA will help us to develop RNaseP and RNaseP protein as targets for novel antibiotics against this pathogen.

Structure-based and combinatorial search for new RNA-binding drugs.

The discovery by structure-based and combinatorial methods of new RNA-binding drugs presents great opportunities for pharmacological development against drug-resistant bacterial and viral pathogens. A handful of recent RNA structures and more numerous studies of the interaction of combinatorial libraries and oligomeric RNA-binding compounds are providing the foundation for effective RNA-targeted drug discovery programs.

The N terminus of eukaryotic translation elongation factor 3 interacts with 18 S rRNA and 80 S ribosomes.

Elongation factor-3 (EF-3) is an essential fungal-specific translation factor which exhibits a strong ribosome-dependent ATPase activity and has sequence homologies that may predict domains critical for its role in protein synthesis, including a domain at the N terminus, which exhibits sequence homology with Escherichia coli ribosomal protein S5. A portion of the N terminus of Saccharomyces cerevisiae EF-3 (spanning the S5 homology region) has been cloned, expressed, and purified from E. coli. UV cross-linking experiments revealed that the N-terminal EF-3 protein (N-term EF-3) can be specifically cross-linked to 18 S rRNA. Filter-binding assays confirmed these data, and also established that the interaction has a Kd approximately 238 nM. Additional evidence shows that N-term EF-3 is able to associate with yeast ribosomes and inhibit the ribosome-dependent ATPase activity of native EF-3. These data taken together suggest that at least one of the ribosome-binding sites of EF-3 is located at the N terminus.

RNA as a drug target.

Historically, the pharmaceutical industry has focused on proteins, rather than nucleic acids, as drug targets. But recent advances in the fields of RNA synthesis, structure determination and therapeutic target identification make the systematic exploitation of RNA as a drug target a realistic goal.

The selection in vivo and characterization of an RNA recognition motif for spectinomycin.

Ribonucleoprotein (RNP) complexes participate in almost all macromolecular processes, including RNA processing, protein synthesis, and the signal recognition of proteins targeted for export. An understanding of these processes requires detailed knowledge of interactions at the molecular level, which has evidently been difficult due to the size and complexity of the particles. Fragmentation of large RNP complexes into functional subdomains is proven to be a successful in vitro strategy to probe ligand interactions at the molecular level. We reasoned that RNA molecules expressed in vivo may fold in such a manner as to mimic a drug binding site present on the intact ribosome. If expressed at sufficient levels, the RNA would sequester the antibiotic thereby permitting the continued function of the ribosome and consequently allow the cell to survive in the presence of the drug. Evidence is presented here in support of this RNA fragment-rescue concept following the selection and characterization of RNA fragments that confer resistance to the antibiotic spectinomycin.

Throwing a spanner in the works: antibiotics and the translation apparatus.

The protein synthetic machinery is essential to all living cells and is one of the major targets for antibiotics. Knowledge of the structure and function of the ribosome and its associated factors is key to understanding the mechanism of drug action. Conversely, drugs have been used as tools to probe the translation cycle, thus providing a means to further our understanding of the steps that lead to protein synthesis. Our current understanding as to how antibiotics disrupt this process is reviewed here, with particular emphasis on the prokaryotic elongation cycle and those drugs that interact with ribosomal RNAs.

Mutations in E.coli 16s rRNA that enhance and decrease the activity of a suppressor tRNA.

The in vivo expression of mutations constructed within helix 34 of 16S rRNA has been examined together with a nonsense tRNA suppressor for their action at stop codons. The data revealed two novel results: in contrast to previous findings, some of the rRNA mutations affected suppression at UAA and UAG nonsense codons. Secondly, both an increase and a decrease in the efficiency of the suppressor tRNA were induced by the mutations. This is the first report that rRNA mutations decreased the efficiency of a suppressor tRNA. The data are interpreted as there being competition between the two release factors (RF-1 and RF-2) for an overlapping domain and that helix 34 influences this interaction.

A rRNA-mRNA base pairing model for UGA-dependent termination.

A series of site-directed mutations has been constructed in E coli 16S rRNA and shown to suppress UGA-dependent translational termination. With the exception of the C726 to G base change, all were constructed in helix 34. Characterization of these mutations is reviewed here and from these data and mRNA-rRNA base pairing model for the termination event is presented. The interaction functions via antiparallel base pairing between either 1 of the 2 UCA motifs in helix 34 and the complementary UGA stop codon on the message, thus forming a quasicontinuous A-type helical structure that is further stabilized by stacking enthalpy. Finally, rRNA motifs potentially required for UAA and UAG-dependent translational termination are discussed.

A single mutation in 16S rRNA that affects mRNA binding and translation-termination.

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.

A single base change at 726 in 16S rRNA radically alters the pattern of proteins synthesized in vivo.

A single base change in 16S rRNA (C-726 to G) was constructed by site-directed mutagenesis and cloned into the multicopy plasmid pKK3535 (generating pKK726G) which contains the complete rrnB operon from Escherichia coli. The mutant 16S rRNA was found predominantly in the 30S subunit fraction but was present in the 70S ribosomes. Protein analyses of the free 30S subunits revealed a decrease in the levels of ribosomal proteins S2 and S21 while the composition of the 70S ribosomes was as the wild-type. Transformants of pKK726G were temperature sensitive for growth, although the mutant ribosomes themselves were translationally active in vivo at 37 and 42 degrees C. Two-dimensional gel electrophoresis of the proteins translated in vivo revealed an altered protein profile which included novel proteins, changes in the levels of normal proteins, and the presence of heat shock proteins (HSPs) at 30 degrees C. Inactivation of the host encoded wild-type ribosomes coincided with a significant decrease in the synthesis of the HSPs. We therefore believe the induction of the HSPs to be a secondary response by the cells to the presence of the abnormal proteins.