ABSTRACT
MOTIVATION: Information about a particular protein or protein family is usually distributed among multiple databases and often in more than one entry in each database. Retrieval and organization of this information can be a laborious task. This task is complicated even further by the existence of alternative terms for the same concept. RESULTS: The PDB, SWISS-PROT, ENZYME, and CATH databases have been imported into a combined relational database, BIOMOLQUEST: A powerful search engine has been built using this database as a back end. The search engine achieves significant improvements in query performance by automatically utilizing cross-references between the legacy databases. The results of the queries are presented in an organized, hierarchical way.
Subject(s)
Databases, Factual , Proteins/chemistry , Proteins/physiology , Abstracting and Indexing , Algorithms , Computational Biology , Database Management Systems , SoftwareABSTRACT
Once a model of the secondary structure of an RNA has been deduced, thermal melting analysis can be used to determine whether the model accounts for all intramolecular interactions of the RNA, or whether noncanonical and tertiary interactions make the structure more stable than predicted, or link parts of the structure in unexpected ways. It is also useful to determine the pH, salt, and temperature ranges under which the RNA adopts a stably folded structure, or to analyze unfolding pathways. This unit discusses sample preparation, instrumentation, and theoretical background. It also provide a sample analysis of tRNA unfolding.
Subject(s)
Biochemistry/methods , Nucleic Acid Conformation , RNA/chemistry , RNA/metabolism , Temperature , Calorimetry , Nucleic Acid Denaturation , RNA, Transfer, Phe/chemistry , RNA, Transfer, Phe/metabolism , Saccharomyces cerevisiae/chemistry , Ultraviolet RaysABSTRACT
Although eukaryotes are not generally sensitive to thiostrepton, growth of the human malaria parasite Plasmodium falciparum is severely inhibited by the drug. The proposed target in P. falciparum is the ribosome of the plastid-like organelle (35 kb circular genome) of unknown function. Positive identification of the drug target would confirm that the organelle is essential for blood-stage development of Plasmodium and help clarify the plastid's biological role. The action of thiostrepton as an antibiotic relates to its affinity for a conserved domain of eubacterial rRNA. Its effect on organelles is unknown. Because a number of different point mutations within the Escherichia coli domain abrogates thiostrepton binding, extensive sequence differences between eubacterial and plastid domains brings into question the site of drug action. We have examined temperature-dependent hyperchromicity profiles of synthetic RNAs corresponding to domains in the plastid and cytoplasmic RNAs of P. falciparum. Thiostrepton induces a tertiary structure in the plastid-like fragment similar to that seen in eubacterial rRNA, even though the two share only about 60% sequence identity. A single point mutation in the plastid-like fragment removes thiostrepton-dependent tertiary structure formation. Thus, the plastid and eubacterial RNAs share a stabilized tertiary structure induced by the drug. This direct indicator of drug sensitivity in eubacteria suggests that the plastid-encoded ribosome is similarly sensitive to thiostrepton and that the plastid is the site of drug action. Correlation of thiostrepton-sensitive and -resistant phenotypes with physical parameters suggests thiostrepton resistance as a selectable marker for plastid transformation.
Subject(s)
Plasmodium falciparum/genetics , RNA, Protozoan/chemistry , RNA, Protozoan/drug effects , Thiostrepton/metabolism , Animals , Base Sequence , Binding Sites , Magnesium/pharmacology , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Plasmodium falciparum/drug effects , Plastids/genetics , Quaternary Ammonium Compounds/pharmacology , RNA, Protozoan/metabolism , RNA, Ribosomal, 23S/chemical synthesis , RNA, Ribosomal, 23S/genetics , Sodium/pharmacology , Substrate Specificity , Thiostrepton/chemistry , Thiostrepton/pharmacologyABSTRACT
A 58 nucleotide fragment of Escherichia coli large subunit ribosomal RNA, nucleotides 1051 to 1108, adopts a specific tertiary structure normally requiring both monovalent (NH4+ or K+) and divalent (Mg2+) ions to fold; this ion-dependent structure is a prerequisite for recognition by ribosomal protein L11. Melting experiments have been used to show that a sequence variant of this fragment, GACG RNA, is able to adopt a stable tertiary structure in the presence of 1.6 M NH4Cl and absence of divalent ions. The similarity of this high-salt structure to the tertiary structure formed under more typical salt conditions (0.1 M NH4Cl and several mM MgCl2) was shown by its following properties: (i) an unusual ratio of hyperchromicity at 260 nm and 280 nm upon unfolding, (ii) selectivity for NH4+ over K+ or Na+, (iii) stabilization by L11 protein, and (iv) further stabilization by added Mg2+. Delocalized electrostatic interactions of divalent ions with nucleic acids should be very weak in the presence of >1 M monovalent salt; thus stabilization of the tertiary structure by low (<1 mM) Mg2+ concentrations in these high-salt conditions suggests that Mg2+ binds at specific site(s). GACG RNA tertiary structure unfolding in 1.6 M NH4Cl (Tm approximately 39 degrees C) is distinct from melting of the secondary structure (centered at approximately 72 degrees C), and it has been possible to calculate the free energy of tertiary structure stabilization upon addition of various divalent cations. From these binding free energies, ion-RNA binding isotherms for Mn2+, Mg2+, Ca2+, Sr2+ and Ba2+ have been obtained. All of these ions bind at two sites: one site favors Mg2+ and Ba2+ and discriminates against Ca2+, while the other site favors binding of smaller ions over larger ones (Mg2+ >Ca2+ >Sr2+ >Ba2+). Weak cooperative or anticooperative interactions between the sites, also dependent on ion radius, may also be taking place.
