ABSTRACT
The solution of a primary 16S rRNA-binding ribosomal protein, S17, was investigated by two- and three-dimensional homonuclear and heteronuclear magnetic resonance spectroscopy. Almost complete chemical shift assignments for the 1H, 15N, and 13C resonances have been obtained. The NMR data have been rigorously analyzed using a combination of distance geometry, back-calculation, and simulated annealing refinement techniques, and a high-resolution three-dimensional structure has been deduced. The protein consists of a single twisted antiparallel beta-pleated sheet with Greek-key topology. The five beta-strands are connected by extended loops that are flexible compared to the beta-sheet core structure and appear not to adopt one definite conformation in solution. Two of these loops contain many of the residues that have been implicated in binding ribosomal RNA. The location and distribution of these residues and other positively charged side chains on the protein surface suggest an interaction with two distinct regions of ribosomal RNA.
Subject(s)
Ribosomal Proteins/chemistry , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Genome, Bacterial , Geobacillus stearothermophilus/genetics , Geobacillus stearothermophilus/metabolism , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Polymerase Chain Reaction , Protein Conformation , Protein Structure, Secondary , RNA, Ribosomal, 16S/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ribosomal Proteins/isolation & purification , Ribosomal Proteins/metabolism , SolutionsABSTRACT
After a long hiatus, the pace of determination of the structures of ribosomal proteins has accelerated dramatically. We discuss here the structures of five ribosomal proteins from Bacillus stearothermophilus: S5, S17, L6, L9, and L14. These structures represent several new motifs. Each of these structures has revealed new insights, and we have developed criteria for recognizing RNA-binding regions of each protein and correlating the structures with such properties as antibiotic resistance. The information here should also prove invaluable in an eventual high-resolution picture of the intact ribosome.
Subject(s)
Bacterial Proteins/chemistry , Biological Evolution , Geobacillus stearothermophilus/chemistry , RNA-Binding Proteins/chemistry , Ribosomal Proteins/chemistry , Bacterial Proteins/metabolism , Protein Structure, Tertiary , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolismABSTRACT
Nine DNA sequences related to the purine strand of the human centromeric satellite (AATGG)n (CCATT)n repeat have been studied by two-dimensional nuclear magnetic resonance spectroscopy. Earlier studies have suggested that the structure of (AATGG)n sequence has an equilibrium between the duplex form and a fold-back form. Structural refinement of d(CAATGG) and its related sequences by an NOE-constrained simulated annealing procedure reveals that the duplex form incorporates dynamic type-I G-A base pairs. 1D exchangeable proton NMR data support this model. The reverse sequence motif (GGTAA) destabilizes the structure.
Subject(s)
Centromere , Chromosomes, Human , DNA, Satellite/chemistry , Repetitive Sequences, Nucleic Acid , Base Composition , Base Sequence , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Nucleic Acid ConformationABSTRACT
The structures of the (C-T)n sequence at two different pHs have been analyzed by 500 MHz 2D-NMR using a modified DNA decamer d(CT[m5C]TCU[m5C]UCT) as a model system. The chemical modifications serve to perturb the monotonous C-T repeat, and consequently to yield a better chemical shift dispersion. The results reinforce our earlier suggestion that there are three major pH-dependent conformational species: two antiparallel-stranded (APS) duplexes at pH 7 and pH 3, and a different structure near pH 5. Structural refinement of the decamer duplexes at pH = 7.5 and pH = 2.9 using 2D-NOE data suggests that the C:T or C+:T base pairs are continuously stacked. Exchangeable proton NMR spectra at pH 7.5 and pH 2.9 are consistent with C:T or C+:T base pairing schemes in which a water molecule bridges the two bases.
Subject(s)
Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , 5-Methylcytosine , Base Composition , Base Sequence , Cytosine/analogs & derivatives , DNA/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Molecular Sequence Data , Oligodeoxyribonucleotides/chemical synthesis , Oligoribonucleotides/chemistry , Polydeoxyribonucleotides/chemistry , ThymineABSTRACT
Alternating (C-T)n sequences are involved in the H-DNA structure associated with (GA)n.(CT)n sequences. Low pH values facilitate H-DNA formation. We have undertaken a detailed analysis of the structural consequences of the (C-T)n sequence as a function of pH. The structures of three DNA oligonucleotides, d(CT)4, d(TC)4 and d(TC)15, have been studied by NMR. We found that their conformations are polymorphic and pH dependent. There are at least three major conformational species: an antiparallel-stranded (APS) duplex with entirely C:T base pairs at pH 7, an antiparallel-stranded (APS) duplex with entirely C+:T base pairs at pH 3, and a possible parallel-stranded (PS) duplex with C+:C and T:T base pairs near pH 5. In the intermediate pH range, the APS duplex may have varying numbers of C+:T and C:T base pairs, and there may be a fast exchange going on between APS duplex species involving these two kinds of base pairs. However, the transition between the APS and PS duplexes is slow. Structural refinement of the two octamers, d(TC)4 and d(CT)4, at pH = 6.9 and pH = 3 using 2D-NOE data suggests that the molecules are likely in the duplex form at 5 degrees C. We lack evidence that the structure at pH 3 is a PS structure with T nucleotides residing in the exterior of the helix. Titration of the longer oligonucleotide, d(TC)15, showed a prominent pKa of approximately 6, approaching the value of 7.0 obtained from the titration of poly-(dC).
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Polymorphism, Genetic , Base Composition , Cytosine , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Protons , ThymidineABSTRACT
RNA and DNA adopt different types of conformations, i.e., A-type with C3'-endo sugar pucker for RNA and B-type with C2'-endo sugar pucker for DNA, respectively. The structural influence of the incorporation of RNA nucleotides into DNA is less understood. In this paper, we present the three-dimensional structures of two RNA-containing oligonucleotides, d(CG)r(CG)d(CG) and d(CG)r(C)d-(TAGCG), as determined by the NMR refinement procedure, and assess the possible structural perturbation of DNA induced by RNA. With a single RNA insertion into an octamer DNA, its overall conformation remains as the canonical B-DNA, except that the sugar pucker of the rC3 residue is C3'-endo (pseudorotation angle P = 3.6 degrees). In contrast, the hybrid hexamer is neither the pure B-DNA nor the pure A-DNA conformation. Instead, we propose a model in which the DNA parts adopt B conformation, whereas the RNA part adopts A conformation, with the overall conformation closer to A-DNA. To ensure an exhaustive search of the conformational space, the model was subjected to 100-ps simulated annealing with slow cooling or 100-ps molecular dynamics with subsequent quenching. Models obtained at different time points of the trajectories were further subjected to the SPEDREF NOE refinement [Robinson & Wang (1992) Biochemistry 31, 3524] and they appeared to arrive at a convergent model (< 0.5 A RMSD for the central four base pairs). The consensus hexamer structure contains a significant discontinuity at the (rG4)p(dC5) step with a base pair tilt angle of 6.7 degrees and roll angle of 11.5 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)
