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David E Draper

Publications and source records attributed to David E Draper.

12 recordsLinked to original sources
PubMed2006

Mg2+-RNA interaction free energies and their relationship to the folding of RNA tertiary structures.

Mg2+ ions are very effective at stabilizing tertiary structures in RNAs. In most cases, folding of an RNA is so strongly coupled to its interactions with Mg2+ that it is difficult to separate free energies of Mg2+-RNA interactions from the intrinsic free energy of RNA folding. To devise quantitative models accounting for this phenomenon of Mg2+-induced RNA folding, it is necessary to independently determine Mg2+-RNA interaction free energies for folded and unfolded RNA forms. In this work, the energetics of Mg2+-RNA interactions are derived from an assay that measures the effective concentration of Mg2+ in the presence of RNA. These measurements are used with other measures of RNA stability to develop an overall picture of the energetics of Mg2+-induced RNA folding. Two different RNAs are discussed, a pseudoknot and an rRNA fragment. Both RNAs interact strongly with Mg2+ when partially unfolded, but the two folded RNAs differ dramatically in their inherent stability in the absence of Mg2+ and in the free energy of their interactions with Mg2+. From these results, it appears that any comprehensive framework for understanding Mg2+-induced stabilization of RNA will have to (i) take into account the interactions of ions with the partially unfolded RNAs and (ii) identify factors responsible for the widely different strengths with which folded tertiary structures interact with Mg2+.

Base Sequence↗
PubMed2006

Optimization of a ribosomal structural domain by natural selection.

A conserved, independently folding domain in the large ribosomal subunit consists of 58 nt of rRNA and a single protein, L11. The tertiary structure of an rRNA fragment carrying the Escherichia coli sequence is marginally stable in vitro but can be substantially stabilized by mutations found in other organisms. To distinguish between possible reasons why natural selection has not evolved a more stable rRNA structure in E. coli, mutations affecting the rRNA tertiary structure were assessed for their in vitro effects on rRNA stability and L11 affinity (in the context of an rRNA fragment) or in vivo effects on cell growth rate and L11 content of ribosomes. The rRNA fragment stabilities ranged from -4 to +9 kcal/mol relative to the wild-type sequence. Variants in the range of -4 to +5 kcal/mol had almost no observable effect in vivo, while more destabilizing mutations (>7 kcal/mol) were not tolerated. The data suggest that the in vivo stability of the complex is roughly -6 kcal/mol and that any single tertiary interaction is dispensable for function as long as a minimum stability of the complex is maintained. On the basis of these data, it seems that the evolution of this domain has not been constrained by inherent structural or functional limits on stability. The estimated stability corresponds to only a few ribosomes per bacterial cell dissociated from L11 at any time; thus the selective advantage for any further increase in stability may be so small as to be outweighed by other competing selective pressures.

Escherichia coli↗
PubMed2005

A small protein unique to bacteria organizes rRNA tertiary structure over an extensive region of the 50 S ribosomal subunit.

A number of small, basic proteins penetrate into the structure of the large subunit of the ribosome. While these proteins presumably aid in the folding of the rRNA, the extent of their contribution to the stability or function of the ribosome is unknown. One of these small, basic proteins is L36, which is highly conserved in Bacteria, but is not present in Archaea or Eucarya. Comparison of ribosome crystal structures shows that the space occupied by L36 in a bacterial ribosome is empty in an archaeal ribosome. To ask what L36 contributes to ribosome stability and function, we have constructed an Escherichia coli strain lacking ribosomal protein L36; cell growth is slowed by 40-50% between 30 degrees C and 42 degrees C. Ribosomes from this deletion strain sediment normally and have a full complement of proteins, other than L36. Chemical protection experiments comparing rRNA from wild-type and L36-deficient ribosomes show the expected increase in reagent accessibility in the immediate vicinity of the L36 binding site, but suggest that a cooperative network of rRNA tertiary interactions has been disrupted along a path extending 60 A deep into the ribosome. These data argue that L36 plays a significant role in organizing 23 S rRNA structure. Perhaps the Archaea and Eucarya have compensated for their lack of L36 by maintaining more stable rRNA tertiary contacts or by adopting alternative protein-RNA interactions elsewhere in the ribosome.

Base Sequence↗
PubMed2005

Interactions of the N-terminal domain of ribosomal protein L11 with thiostrepton and rRNA.

Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2'-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives approximately 15-fold enhancement when L11 or L11-C76 is bound. Although the pyrene tag weakens protein binding, unbiased protein-RNA association constants were obtained in competition experiments with untagged RNA. It was found that (i) intact B. stearothermophilus L11 binds rRNA with K approximately 1.2 x 10(9) m(-1) in buffers with 0.2 m KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (approximately 8-fold enhancement at 0.2 m KCl), (iii) L11 stimulates thiostrepton binding by 2.3 +/- 0.6 x 10(3)-fold, predicting an overall thiostrepton affinity for the ribosome of approximately 10(9) m(-1), and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding. The latter observation resolves the question of why eukaryotes are insensitive to the antibiotic. These measurements also show that it is plausible for thiostrepton to compete directly with EF-G.GDP for binding to the L11-RNA complex, and provide a quantitative basis for further studies of L11 function and thiostrepton mechanism.

Anti-Bacterial Agents↗
PubMed2005

Coevolution of protein and RNA structures within a highly conserved ribosomal domain.

The X-ray crystal structure of a ribosomal L11-rRNA complex with chloroplast-like mutations in both protein and rRNA is presented. The global structure is almost identical to that of the wild-type (bacterial) complex, with only a small movement of the protein alpha helix away from the surface of the RNA required to accommodate the altered protein residue. In contrast, the specific hydrogen bonding pattern of the mutated residues is substantially different, and now includes a direct interaction between the protein side chain and an RNA base edge and a water-mediated contact. Comparison of the two structures allows the observations of sequence variation and relative affinities of wild-type and mutant complexes to be clearly rationalized, but reinforces the concept that there is no single simple code for protein-RNA recognition.

Base Sequence↗
PubMed2005

Ions and RNA folding.

The problem of how ions influence the folding of RNA into specific tertiary structures is being addressed from both thermodynamic (by how much do different salts affect the free energy change of folding) and structural (how are ions arranged on or near an RNA and what kinds of environments do they occupy) points of view. The challenge is to link these different approaches in a theoretical framework that relates the energetics of ion-RNA interactions to the spatial distribution of ions. This review distinguishes three different kinds of ion environments that differ in the extent of direct ion-RNA contacts and the degree to which the ion hydration is perturbed, and summarizes the current understanding of the way each environment relates to the overall energetics of RNA folding.

Biophysics↗
PubMed2004

A guide to ions and RNA structure.

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. "Diffuse ions" accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A "chelated" ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg(2+) on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.

Animals↗
PubMed2003

Electrostatic interactions in a peptide--RNA complex.

Parallel experimental measurements and theoretical calculations have been used to investigate the energetics of electrostatic interactions in the complex formed between a 22 residue, alpha-helical peptide from the N protein of phage lambda and its cognate 19 nucleotide box B RNA hairpin. Salt-dependent free energies were measured for both peptide folding from coil to helix and peptide binding to RNA, and from these the salt-dependence of binding pre-folded, helical peptide to RNA was determined ( partial differential (DeltaG degrees (dock))/ partial differential log[KCl]=5.98(+/-0.21)kcal/mol). (A folding transition taking place in the RNA hairpin loop was shown to have a negligible dependence on salt concentration.) The non-linear Poisson-Boltzmann equation was used to calculate the same salt dependence of the binding free energy as 5.87(+/-0.22)kcal/mol, in excellent agreement with the measured value. Close agreement between experimental measurements and calculations was also obtained for two variant peptides in which either a basic or acidic residue was replaced with an uncharged residue, and for an RNA variant with a deletion of a single loop nucleotide. The calculations suggest that the strength of electrostatic interactions between a peptide residue and RNA varies considerably with environment, but that all 12 positive and negative N peptide charges contribute significantly to the electrostatic free energy of RNA binding, even at distances up to 11A from backbone phosphate groups. Calculations also show that the net release of ions that accompanies complex formation originates from rearrangements of both peptide and RNA ion atmospheres, and includes accumulation of ions in some regions of the complex as well as displacement of cations and anions from the ion atmospheres of the RNA and peptide, respectively.

Amino Acid Sequence↗
PubMed2003

A thermodynamic framework for the magnesium-dependent folding of RNA.

The goal of this review is to present a unified picture of the relationship between ion binding and RNA folding based on recent theoretical and computational advances. In particular, we present a model describing how the association of magnesium ions is coupled to the tertiary structure folding of several well-characterized RNA molecules. This model is developed in terms of the nonlinear Poisson-Boltzmann (NLPB) equation, which provides a rigorous electrostatic description of the interaction between Mg(2+) and specific RNA structures. In our description, most of the ions surrounding an RNA behave as a thermally fluctuating ensemble distributed according to a Boltzmann weighted average of the mean electrostatic potential around the RNA. In some cases, however, individual ions near the RNA may shed some of their surrounding waters to optimize their Coulombic interactions with the negatively charged ligands on the RNA. These chelated ions are energetically distinct from the surrounding ensemble and must be treated explicitly. This model is used to explore several different RNA systems that interact differently with Mg(2+). In each case, the NLPB equation accurately describes the stoichiometric and energetic linkage between Mg(2+) binding and RNA folding without requiring any fitted parameters in the calculation. Based on this model, we present a physical description of how Mg(2+) binds and stabilizes specific RNA structures to promote the folding reaction.

Binding Sites↗
PubMed2002

A compact RNA tertiary structure contains a buried backbone-K+ complex.

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.

Base Sequence↗
PubMed2002

The linkage between magnesium binding and RNA folding.

Understanding the linkage between Mg(2+) binding and RNA folding requires a proper theoretical model describing the energetics of Mg(2+) binding to the folded and unfolded states of RNA. Our current understanding of Mg(2+) binding to these different RNA states derives from empirical thermodynamic models that depend on a number of unjustified assumptions. We present a rigorous theoretical model describing the linkage between RNA folding and magnesium ion binding. In this model, based on the non-linear Poisson-Boltzmann (NLPB) equation, the stabilization of RNA by Mg(2+) arises from two distinct binding modes, diffuse binding and site binding. Diffusely bound Mg(2+) are described as an ensemble of hydrated ions that are attracted to the negative charge of the RNA. Site-bound Mg(2+) are partially desolvated ions that are attracted to electronegative pockets on the RNA surface. We explore two systems, yeast tRNA(Phe) and a 58-nucleotide rRNA fragment, with different Mg(2+) binding properties. The NLPB equation accurately describes both the stoichiometric and energetic linkage between Mg(2+) binding and RNA folding for both of these systems without requiring any fitted parameters in the calculation. Moreover, the NLPB model presents a well-defined physical description of how Mg(2+) binding helps fold an RNA. For both of the molecules studied here, the relevant unfolded state is a disordered intermediate state (I) that contains stable helical secondary structure without any tertiary contacts. Diffusely bound Mg(2+) interact with these secondary structure elements to stabilize the I state. The secondary structural elements of the I state fold into a compact, native tertiary structure (the N state). Diffuse binding plays a dominant role in stabilizing the N state for both RNAs studied. However, for the rRNA fragment, site-binding to a location with extraordinarily high electrostatic potential is also coupled to folding. Our results suggest that much experimental data measuring the linkage between Mg(2+) binding and RNA folding must be reinterpreted.

Escherichia coli↗