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D E Draper

Publications and source records attributed to D E Draper.

At least 19 recordsLinked to original sources
PubMed1998

A functional ribosomal RNA tertiary structure involves a base triple interaction.

Comparative sequence analysis reveals a coordinated set of nucleotide exchanges between the base pair 1092/1099 and the unpaired position 1072 [(1092/1099)1072] in the L11 binding domain of 23S ribosomal RNA. This set of exchanges has occurred at least 4 times during evolution, suggesting that these positions form a base triple. The analysis further suggests an important role for positions (1065/1073), adjacent to 1072. The covariation at positions (1092/1099)1072 is studied here by analysis of RNA variants using UV melting and binding of ribosomal protein L11 and thiostrepton to assay for tertiary folding of this domain. The tertiary structure of the RNA is eliminated by alteration of the unpaired nucleotide (C1072 to U mutation), and binding of L11 and thiostrepton are reduced 10-fold compared to the wild type. In contrast, substitution of the base pair (CG1092/1099 to UA mutation) allows formation of the tertiary structure but dramatically alters the pH dependence of tertiary folding. The fully compensated set of mutations, (CG)C to (UA)U, restores the tertiary structure of the RNA to a state almost identical to the wild type. The nature of this base triple and its implications for the folding of the RNA and ligand interactions are discussed.

Base Composition↗
PubMed1998

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif.

We report the 1.7 A crystal structure of ribosomal protein S4 from Bacillus stearothermophilus. To facilitate the crystallization, 41 apparently flexible residues at the N-terminus of the protein have been deleted (S4Delta41). S4Delta41 has two domains; domain 1 is completely alpha-helical and domain 2 comprises a five-stranded antiparallel beta-sheet with three alpha-helices packed on one side. Domain 2 is an insertion within domain 1, and it shows significant structural homology to the ETS domain of eukaryotic transcription factors. A phylogenetic analysis of the S4 primary structure shows that the likely RNA interaction surface is predominantly on one side of the protein. The surface is extensive and highly positively charged, and is centered on a distinctive canyon at the domain interface. The latter feature contains two arginines that are totally conserved in all known species of S4 including eukaryotes, and are probably crucial in binding RNA. As has been shown for other ribosomal proteins, mutations within S4 that affect ribosome function appear to disrupt the RNA-binding sites. The structure provides a framework with which to probe the RNA-binding properties of S4 by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1998

The solution structure of ribosomal protein S4 delta41 reveals two subdomains and a positively charged surface that may interact with RNA.

S4 is one of the first proteins to bind to 16S RNA during assembly of the prokaryotic ribosome. Residues 43-200 of S4 from Bacillus stearothermophilus (S4 Delta41) bind specifically to both 16S rRNA and to a pseudoknot within the alpha operon mRNA. As a first step toward understanding how S4 recognizes and organizes RNA, we have solved the structure of S4 Delta41 in solution by multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The fold consists of two globular subdomains, one comprised of four helices and the other comprised of a five-stranded antiparallel beta-sheet and three helices. Although cross-linking studies suggest that residues between helices alpha2 and alpha3 are close to RNA, the concentration of positive charge along the crevice between the two subdomains suggests that this could be an RNA-binding site. In contrast to the L11 RNA-binding domain studied previously, S4 Delta41 shows no fast local motions, suggesting that it has less capacity for refolding to fit RNA. The independently determined crystal structure of S4 Delta41 shows similar features, although there is small rotation of the subdomains compared with the solution structure. The relative orientation of the subdomains in solution will be verified with further study.

Amino Acid Sequence↗
PubMed1998

RNA structure.

New information concerning RNA structure is accumulating at an ever increasing rate-from short helices with mismatched bases of 5S rRNA and complex RNA aptamers. The importance of recurring structural motifs, ion binding, and the kinetics and energetics of folding in RNA structure and function is now being recognized and addressed.

Animals↗
PubMed1997

Folding of an mRNA pseudoknot required for stop codon readthrough: effects of mono- and divalent ions on stability.

Unfolding of an mRNA pseudoknot that induces ribosome suppression of the gag gene stop codon in Moloney murine leukemia virus has been studied by UV hyperchromicity and calorimetry. The pseudoknot melts in two steps, corresponding to its two helical stems. The total enthalpy of denaturation is approximately 170 kcal/mol, approximately the value expected for the secondary structure. At low salt concentrations (<50 mM KCl) the unfolding transitions are not two-state, but they approach two-state behavior at higher salt concentrations. The structure is preferentially stabilized by smaller alkali metal ions (Li+ > Na+ > K+ > Rb+ > Cs+) and by NH4+; the same preferences are exhibited by one of the stems in the context of a hairpin. Divalent metal ions are not required to fold the pseudoknot but do stabilize it further. To examine divalent ion effects over a wide concentration range, urea was used to lower the RNA unfolding temperature and was shown not to affect characteristics of the pseudoknot unfolding in other respects. The pseudoknot binds divalent ions somewhat more tightly than a hairpin but shows only weak selectivity for different size ions. It is suggested that a region of "intermediate" divalent ion binding affinity, in between highly ligated specific sites and purely delocalized ion binding in character, is created by the pseudoknot fold but that nonspecific, delocalized ion binding contributes at least half the free energy of pseudoknot stabilization by Mg2+.

Animals↗
PubMed1997

The RNA binding domain of ribosomal protein L11: three-dimensional structure of the RNA-bound form of the protein and its interaction with 23 S rRNA.

The three-dimensional solution structure has been determined by NMR spectroscopy of the 75 residue C-terminal domain of ribosomal protein L11 (L11-C76) in its RNA-bound state. L11-C76 recognizes and binds tightly to a highly conserved 58 nucleotide domain of 23 S ribosomal RNA, whose secondary structure consists of three helical stems and a central junction loop. The NMR data reveal that the conserved structural core of the protein, which consists of a bundle of three alpha-helices and a two-stranded parallel beta-sheet four residues in length, is nearly the same as the solution structure determined for the non-liganded form of the protein. There are however, substantial chemical shift perturbations which accompany RNA binding, the largest of which map onto an extended loop which bridges the C-terminal end of alpha-helix 1 and the first strand of parallel beta-sheet. Substantial shift perturbations are also observed in the N-terminal end of alpha-helix 1, the intervening loop that bridges helices 2 and 3, and alpha-helix 3. The four contact regions identified by the shift perturbation data also displayed protein-RNA NOEs, as identified by isotope-filtered three-dimensional NOE spectroscopy. The shift perturbation and NOE data not only implicate helix 3 as playing an important role in RNA binding, but also indicate that regions flanking helix 3 are involved as well. Loop 1 is of particular interest as it was found to be flexible and disordered for L11-C76 free in solution, but not in the RNA-bound form of the protein, where it appears rigid and adopts a specific conformation as a result of its direct contact to RNA.

Amino Acid Sequence↗
PubMed1997

Affinities and selectivities of divalent cation binding sites within an RNA tertiary structure.

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.

Barium↗
PubMed1997

Effects of Mg2+, K+, and H+ on an equilibrium between alternative conformations of an RNA pseudoknot.

A complex pseudoknot structure surrounds the first ribosome initiation site in the Escherichia coli alpha mRNA and mediates its regulation by ribosomal protein S4. A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature. Between 30 degrees C and 45 degrees C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H+, K+ and Mg2+ concentrations. Mg2+ favors formation of the "fast" electrophoretic form: a single Mg2+ is bound in the rate-limiting step, followed by cooperative binding of approximately 1.7 additional ions. Binding of the latter ions provides most of the favorable free energy for the reaction. However, the "slow" form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg2+ concentrations drive the equilibrium to only approximatley 70% fast form. A single H+ is taken up in the switch to the "slow" conformer, which has apparent pK approximately 5.9; low pH also stabilizes part of the pseudoknot structure melting at approximately 62 degrees C. Mg2+ and H+ appear to direct alpha mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers. K+ has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers. The alpha mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step.

Base Sequence↗
PubMed1997

In vitro evolution used to define a protein recognition site within a large RNA domain.

A minimum of 460 nucleotides of 16S ribosomal RNA are needed to fold the target site for E. coli ribosomal protein S4, although a much smaller region within this large domain is protected from chemical reagents by the protein. Starting with a 531-nucleotide tRNA fragment, cycles of mutagenesis, selection with S4, and amplification ('in vitro evolution') were used to obtain a pool of 30 RNA sequences selected for S4 recognition but approximately 30% different from wild type. Numerous compensatory base pair changes have largely preserved the same secondary structure among these RNAs as found in wild-type sequences. A 20-base deletion and a single nucleotide insertion are among several unusual features found in most of the selected sequences and also prevalent among other prokaryotic rRNAs. Most of the compensatory base changes and selected features are located outside of the region protected by S4 from chemical reagents. It was unexpected that S4 would select for RNA structures throughout such a large domain; the selected features are probably contributing indirectly to S4 recognition by promoting correct tertiary folding of the region actually contacted by S4. The role of S4 may be to stabilize this domain (nearly one-third of the 16S rRNA) in its proper conformation for ribosome function.

Base Sequence↗
PubMed1997

High resolution solution structure of ribosomal protein L11-C76, a helical protein with a flexible loop that becomes structured upon binding to RNA.

The structure of the C-terminal RNA recognition domain of ribosomal protein L11 has been solved by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy. Although the structure can be considered high resolution in the core, 15 residues between helix alpha 1 and strand beta 1 form an extended, unstructured loop. 15N transverse relaxation measurements suggest that the loop is moving on a picosecond-to-nanosecond time scale in the free protein but not in the protein bound to RNA. Chemical shifts differences between the free protein and the bound protein suggest that the loop as well as the C-terminal end of helix alpha 3 are involved in RNA binding.

Amino Acid Sequence↗
PubMed1997

Interaction of thiostrepton with an RNA fragment derived from the plastid-encoded ribosomal RNA of the malaria parasite.

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.

Animals↗
PubMed1996

Structure of a U.U pair within a conserved ribosomal RNA hairpin.

A conserved hairpin corresponding to nt 1057-1081 of large subunit rRNA (Escherichia coli numbering) is part of a domain targeted by antibiotics and ribosomal protein L11. The stem of the hairpin contains a U.U juxtaposition, found as either U.U or U.C in virtually all rRNA sequences. This hairpin has been synthesized and most of the aromatic and sugar protons were assigned by two-dimensional proton NMR. Distances and sugar puckers deduced from the NMR data were combined with restrained molecular dynamics calculations to deduce structural features of the hairpin. The two U residues are stacked in the helix, form one NH3-O4 hydrogen bond and require an extended backbone conformation (trans alpha and gamma) at one of the U nucleotides. The hairpin loop, UAGAAGC closed by a U-A pair, is the same size as tRNA anticodon loops, but not as well ordered.

Anticodon↗
PubMed1996

Structure of a hexanucleotide RNA hairpin loop conserved in ribosomal RNAs.

The structure of 5'-pppGGAC(GUAAUA)GUCC has been deduced from NMR data. The six-nucleotide hairpin loop is highly conserved in large subunit ribosomal RNA and confers unusual stability on RNA hairpins. Standard assignment methods, including through-bond strategies for backbone protons, were used to assign all 31P resonances and all but two of the non-exchangeable protons. The model calculated by restrained molecular dynamics shows that the loop adopts a specific structure stabilized by five non-canonical hydrogen bonds. An edge-to-edge G-A pair (hydrogen bonds G5 N3-A10 NH6, G5 H2-A1O N7 and A10 NH6-G5 O2') closes the hairpin loop. A trans 5' P-O bond at A7 reverses the direction of the backbone and is stabilized by two base-backbone hydrogen bonds (U6 NH3-U9 OP and U6 O2'-A8 N7). The only unstructured part of the loop is U9, which is completely unstacked and also the only phylogenetically variable position. The U6-A7-A8 "U-turn" reproduces hydrogen bonds and backbone torsion seen in the tRNA anticodon loop and TTpsiC loop, and in an internal loop of the hammerhead ribozyme. G-A is a common closing mismatch in ribosomal RNA hairpin loops. The U-turn and G-A pair found in this hexaloop structure may therefore be common structural units of RNA hairpin loops.

Base Sequence↗
PubMed1996

Cooperative interactions of RNA and thiostrepton antibiotic with two domains of ribosomal protein L11.

Ribosomal protein L11 interacts with a 58-nucleotide domain of large subunit ribosomal RNA; both the protein and its RNA target have been highly conserved. The antibiotic thiostrepton recognizes the same RNA domain, and binds to the ribosome cooperatively with L11. Experiments presented here show that RNA recognition and thiostrepton cooperativity can be attributed to C- and N-terminal domains of L11, respectively. Under trypsin digestion conditions that degrade Bacillus stearothermophilus L11 to small fragments, the target RNA protects the C-terminal 77 residues from digestion, and thiostrepton and RNA in combination protect the entire protein. A 76-residue C-terminal fragment of L11 was overexpressed and shown to fold into a stable structure binding ribosomal RNA with essentially the same properties as full-length L11. An L11.thiostrepton.RNA complex was 100-200-fold more stable than expected on the basis of L11-RNA and thiostrepton-RNA binding affinities; similar measurements with the C-terminal fragment detected no cooperativity with thiostrepton. L11 function is thus more complex than simple interaction with ribosomal RNA; we suggest that thiostrepton mimics some ribosomal component or factor that normally interacts with the L11 N-terminal domain.

Anti-Bacterial Agents↗
PubMed1996

Parallel worlds.

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Animals↗
PubMed1996

Strategies for RNA folding.

RNAs are surprisingly adept at folding into specific shapes capable of ligand recognition and catalysis. Thermodynamic analysis of the unfolding of several different RNAs suggests that there are at least three strategies an RNA might use to achieve a very stable and compactly folded structure: hydrogen bonding between irregular complementary surfaces (as in transfer RNA tertiary structure); monovalent and divalent lons bound to specific sites (as found in a ribosomal RNA fragment) and pseudoknot folds (exemplified by a messenger RNA fragment with extensive non-canonical structure).

Nucleic Acid Conformation↗
PubMed1995

Messenger RNA recognition by fragments of ribosomal protein S4.

Ribosomal protein S4 from Escherichia coli binds a large domain of 16 S ribosomal RNA and also a pseudoknot structure in the alpha operon mRNA, where it represses its own synthesis. No similarity between the two RNA binding sites has been detected. To find out whether separate protein regions are responsible for rRNA and mRNA recognition, proteins with N-terminal or C-terminal deletions have been overexpressed and purified. Protein-mRNA interactions were detected by (i) a nitrocellulose filter binding assay, (ii) inhibition of primer extension by reverse transcriptase, and (iii) a gel shift assay. Circular dichroism spectra were taken to determine whether the proteins adopted stable secondary structures. From these studies it is concluded that amino acids 48-104 make specific contacts with the mRNA, although residues 105-177 (out of 205) are required to observe the same toeprint pattern as full-length protein and may stabilize a specific portion of the mRNA structure. These results parallel ribosomal RNA binding properties of similar fragments (Conrad, R. C., and Craven, G. R. (1987) Nucleic Acids Res. 15, 10331-10343, and references therein). It appears that the same protein domain is responsible for both mRNA and rRNA binding activities.

Amino Acid Sequence↗