ABSTRACT
Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus. The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way junctions at both ends composed of portions of helices 20, 21, and 23. Addition of the remaining portion of helix 23 to this construct results in the minimal site for S11. Surprisingly, almost half of the central domain (helices 24, 25, and 26) is dispensable for binding the central domain proteins. Thus, at least two classes of RNA elements can be identified in ribosomal RNA. A protein-binding core element (such as helices 20, 21, 22, and 23) is required for the association of ribosomal proteins, whereas secondary binding elements (such as helices 24, 25, and 26) associate only with the preformed core RNP complex. Apparently, there may be a hierarchy of ribosomal RNA elements similar to the hierarchy of primary, secondary, and tertiary binding ribosomal proteins.
Subject(s)
RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Base Sequence , Binding Sites , Chemical Fractionation , Molecular Sequence Data , Nucleic Acid Conformation , RNA Processing, Post-Transcriptional , Sequence Deletion , Thermus thermophilus/geneticsABSTRACT
The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.
Subject(s)
RNA, Ribosomal/chemistry , Ribonucleoproteins/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Thermus thermophilus/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Pairing , Base Sequence , Binding Sites , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal/metabolism , Ribonucleoproteins/metabolism , Ribosomal Protein S6 , Ribosomal Proteins/metabolism , Thermus thermophilus/ultrastructureABSTRACT
Fragments of the 16S rRNA of Thermus thermophilus representing the 3' domain (nucleotides 890-1515) and the 5' domain (nucleotides 1-539) have been prepared by transcription in vitro. Incubation of these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3' RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3' domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5' RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit [Agalarov, S.C., Zheleznyakova, E.N., Selivanova, O.M., Zheleznaya, L.A., Matvienko, N.I., Vasiliev, V.D. & Spirin, A.S. (1998) Proc. Natl Acad. Sci. USA 95, 999-1003], these experiments establish that all three main structural lobes of the small ribosomal subunit can be reconstituted independently of each other and prepared in the individual state.
Subject(s)
Ribosomal Proteins/chemistry , Thermus thermophilus/chemistry , Centrifugation, Density Gradient , Electrophoresis, Gel, Two-Dimensional , Nucleic Acid Conformation , Plasmids/metabolism , Protein Structure, Tertiary , RNA/chemistry , Sucrose/pharmacology , Thermus thermophilus/metabolism , Transcription, GeneticABSTRACT
A fragment of the 16S RNA of Thermus thermophilus corresponding to the central domain (nucleotides 547-895) has been prepared by transcription in vitro. Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle. This particle contained five T. thermophilus proteins corresponding to Escherichia coli ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit. When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit. We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.
Subject(s)
RNA, Bacterial/chemistry , RNA, Ribosomal, 16S/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/genetics , Base Sequence , Escherichia coli , Microscopy, Electron , Molecular Sequence Data , Nucleic Acid Conformation , Protein Folding , RNA, Bacterial/ultrastructure , RNA, Ribosomal/chemistry , RNA, Ribosomal/ultrastructure , RNA, Ribosomal, 16S/ultrastructure , Ribosomal Proteins/ultrastructure , Thermus thermophilus/ultrastructure , Transcription, GeneticABSTRACT
In parallel with crystallographic studies of ribosomes from Thermus thermophilus, a long-term program on the crystallization and structural investigations of ribosomal proteins from the same microorganism has been started at the Institute of Protein Research (Pushchino, Russia). At present, more than half of the individual ribosomal proteins from T thermophilus have been purified without denaturating agents on a preparative scale and some of them have been obtained in the crystalline form. X-ray structural analysis of two ribosomal proteins, L1 and S6, is being carried out jointly with the Institute of Molecular Biology (Moscow, Russia) and laboratory of professor A Liljas (Lund University, Sweden). L1 is the large protein of the large ribosomal subunit. It can bind not only to a specific site on the 23S rRNA, but also to the mRNA that codes for L1 and L11, thereby acting as a translational repressor for the synthesis of these proteins. The crystals of L1 are orthorhombic and diffract to about 2 A resolution. Native data and data for several heavy atom derivatives have been collected. S6 is a small acidic protein from the small ribosomal subunit. The crystals of S6 are orthorhombic and diffract to 2 A resolution. Native data and derivatives' data have been collected.
Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/chemistry , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Crystallization , Molecular Sequence Data , Ribosomal Proteins/isolation & purificationABSTRACT
Crystals have been obtained of protein L1 from the large ribosomal subunit of an extreme thermophile. Thermus thermophilus, using a mixed solution of ammonium sulphate/methane pentanediol. The crystals belong to the space group P2(1)2(1)2, with cell parameters a = 82.7 A, b = 63.4 A, c = 44.7 A. They diffract X-rays to 2.3 A resolution.
Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Thermus/analysis , X-Ray DiffractionABSTRACT
X-ray scattering, neutron scattering and velocity sedimentation techniques were used for studies of ribosomal 16 S RNA in the isolated state and in different complexes with ribosomal proteins. The neutron scattering curve of the ribosomal 30 S subparticle in 42% 2H2O where the protein component is contrast-matched, was taken as a standard of comparison characterizing the dimensions and shape of the 16 S RNA in situ. The following deductions result from the comparisons. The shape of the isolated 16 S RNA at a sufficient Mg2+ concentration (e.g., in the reconstruction buffer) is similar to that of the 16 S RNA in situ, i.e. in the 30 S particle, but it is somewhat less compact. The 16 S RNA in the complex with protein S4 has a shape and compactness similar to those of the isolated 16 S RNA. The 16 S RNA in the complex with four core proteins, namely S4, S7, S8 and S15, has a shape and compactness similar to those of the isolated 16 S RNA. The six ribosomal proteins S4, S7, S8, S15, S16 and S17 are necessary and sufficient for the 16 S RNA to acquire a compactness similar to that within the 30 S particle. The general conclusion is that the overall specific folding of the 16 S RNA is governed and maintained by its own intramolecular interactions, but the additional folding-up (about one-fourth of the linear size of the whole molecule) or the stabilization of the final compactness requires some ribosomal proteins.
Subject(s)
RNA, Ribosomal , Ribosomal Proteins , Electrophoresis, Polyacrylamide Gel , Neutrons , Nucleic Acid Conformation , Protein Conformation , Scattering, Radiation , UltracentrifugationABSTRACT
A detailed comparative analysis of tryptophan fluorescence spectra of 'sodium' and 'potassium' forms of (Na+, K+)-activated ATPase was carried out. The 'potassium' form spectrum is shifted relative to that of the 'sodium' form by approximately 0.5-1 nm towards shorter wavelengths. The maximal amplitude of the difference spectrum for these forms makes up about 2% of maximal fluorescence intensity of any of the forms. The shape of the difference spectrum does not depend on the solution temperature or ionic strength. The spectral differences between the forms are reversible upon addition of a functionally opposite cation (K+ for 'sodium' form and vice versa) into the medium. The results suggest that if the differences in fluorescence spectra of the 'sodium' and 'potassium' forms of (Na+, K+)-ATPase resulted from the differences in the protein structure, they may be caused by an alteration in local environment of no more than one or two tryptophan residues.
