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D E Draper

Publications and source records attributed to D E Draper.

At least 73 records · Page 4Linked to original sources
PubMed1991

Recognition of the highly conserved GTPase center of 23 S ribosomal RNA by ribosomal protein L11 and the antibiotic thiostrepton.

The antibiotic thiostrepton, a thiazole-containing peptide, inhibits translation and ribosomal GTPase activity by binding directly to a limited and highly conserved region of the large subunit ribosomal RNA termed the GTPase center. We have previously used a filter binding assay to examine the binding of ribosomal protein L11 to a set of ribosomal RNA fragments encompassing the Escherichia coli GTPase center sequence. We show here that thiostrepton binding to the same RNA fragments can also be detected in a filter binding assay. Binding is relatively independent of monovalent salt concentration and temperature but requires a minimum Mg2+ concentration of about 0.5 mM. To help determine the RNA features recognized by L11 and thiostrepton, a set of over 40 RNA sequence variants was prepared which, taken together, change every nucleotide within the 1051 to 1108 recognition domain while preserving the known secondary structure of the RNA. Binding constants for L11 and thiostrepton interaction with these RNAs were measured. Only a small number of sequence variants had more than fivefold effects on L11 binding affinities, and most of these were clustered around a junction of helical segments. These same mutants had similar effects on thiostrepton binding, but more than half of the other sequence changes substantially reduced thiostrepton binding. On the basis of these data and chemical modification studies of this RNA domain in the literature, we propose that L11 makes few, if any, contacts with RNA bases, but recognizes the three-dimensional conformation of the RNA backbone. We also argue from the data that thiostrepton is probably sensitive to small changes in RNA conformation. The results are discussed in terms of a model in which conformational flexibility of the GTPase center RNA is functionally important during the ribosome elongation cycle.

Base Composition↗
PubMed1991

Detection of a key tertiary interaction in the highly conserved GTPase center of large subunit ribosomal RNA.

Searches of ribosomal RNA sequences for compensatory base changes preserving Watson-Crick base pairing have led to detailed models of the conserved secondary structures of these RNAs. In principle, tertiary interactions can also be detected by searches for phylogenetically covariant bases. Within a highly conserved region of the large subunit ribosomal RNA termed the "GTPase center," the bases G-1056-U-1082.A-1086 are found in all eubacteria (Escherichia coli numbering), while A-1056.C-1082.G-1086 are found at the homologous positions in eukaryotes; archaebacteria fall into either category with some exceptions. Either sequence can potentially form a similar set of hydrogen bonds connecting the 3 bases. To determine the contribution of these 3 bases to RNA tertiary structure, sequence variants were made in RNA fragments covering the GTPase center. Correct folding of the RNA fragments was assayed by measuring the binding affinities of two different ligands that recognize the RNA tertiary structure: the highly conserved ribosomal protein L11, which is normally associated with the GTPase center RNA, and the peptide antibiotic thiostrepton, which inhibits the GTPase activity of eubacterial and some archaebacterial ribosomes. The results strongly support the existence of a base pair between positions 1082 and 1086: single mutations at either position weaken both L11 and thiostrepton binding by approximately 10-fold or more, while compensatory double mutations bind the ligands nearly as well as the wild-type E. coli sequence. Variants at position 1056 have little effect on either L11 or thiostrepton binding; a 3-base interaction is therefore not supported by these experiments. A base pair between positions 1082 and 1086 strongly constrains the geometry with which three helical segments join in the middle of the GTPase center.

Base Sequence↗
PubMed1990

Dissection of the 16S rRNA binding site for ribosomal protein S4.

The ribosomal protein S4 from Escherichia coli is essential for initiation of assembly of 30S ribosomal subunits. We have undertaken the identification of specific features required in the 16S rRNA for S4 recognition by synthesizing mutants bearing deletions within a 460 nucleotide region which contains the minimum S4 binding site. We made a set of large nested deletions in a subdomain of the molecule, as well as individual deletions of nine hairpins, and used a nitrocellulose filter binding assay to calculate association constants. Some small hairpins can be eliminated with only minor effects on S4 recognition, while three hairpins scattered throughout the domain (76-90, 376-389 and 456-476) are essential for specific interaction. The loop sequence of hairpin 456-476 is important for S4 binding, and may be directly recognized by the protein. Some of the essential features are in phylogenetically variable regions; consistent with this, Mycoplasma capricolum rRNA is only weakly recognized by S4, and no specific binding to Xenopus laevis rRNA can be detected.

Animals↗
PubMed1990

Bulge loops used to measure the helical twist of RNA in solution.

Bulge loops are commonly found in helical segments of cellular RNAs. When incorporated into long double-stranded RNAs, they may introduce points of flexibility or permanent bend that can be detected by the altered electrophoretic gel mobility of the RNA. We find that a single An or Un bulge loop near the middle of a long RNA helix significantly retards the RNA during polyacrylamide gel electrophoresis if n greater than or equal to 2. The mobility of an RNA containing two A2 bulges various periodically with the number of base pairs between the bulges. We interpret this to mean that A2 bulges varies periodically with the number of base pairs between the bulges. We interpret this to mean that Z2 bulges form torsionally stiff bends in the helix; the gel mobility reaches a minimum when the total helical twist between the bulges rotates the arms of the molecule into a cis conformation. The gel mobilities are proportional to the predicted end-to-end distance of the RNA if the average RNA helical repeat is 11.8 +/- 0.2 bp/turn and there is no helical twist (3 +/- 9 degrees) associated with the bulge (data obtained in 0.15 M Na+). Other sizes and sequences of bulges have very different effects on RNA helix conformation and flexibility. U2 bulges bend the helix to a much smaller degree than A2 bulges, while longer A or U bulge sequences probably allow bends of 90 degrees or more; all of these may be fairly flexible joints.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA.

Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon. In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) [Tang, C. K., & Draper, D. E. (1989) Cell 57, 531]. We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced. Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured. Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression. An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly. Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding. We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled. Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding. This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.

Allosteric Regulation↗
PubMed1989

Thermodynamics of protein-RNA recognition in a highly conserved region of the large-subunit ribosomal RNA.

Ribosomal protein L11 from Escherichia coli specifically binds to a highly conserved region of 23S ribosomal RNA. The thermodynamics of forming a complex between this protein and several different rRNA fragments have been investigated, by use of a nitrocellulose filter binding assay. A 57-nucleotide region of the RNA (C1052-U1108) contains all the protein recognition features, and an RNA fragment containing this region binds L11 10(3)-10(4)-fold more tightly than tRNA. Binding constants are on the order of 10 microM-1 and are only weakly dependent on K+ concentration (delta log K/delta log [K+] = -1.4) or temperature. Binding requires multivalent cations; Mg2+ is taken up into the complex with an affinity of approximately 3 mM-1. Other multivalent cations tested, Ca2+ and Co(NH3)63+, promote binding nearly as well. The pH dependence of binding is a bell-shaped curve with a maximum near neutral pH, but the entire curve is shifted to higher pH for the smaller of two RNA fragments tested. This result suggests that the smaller fragment favors a conformation stabilizing protonated forms of the RNA recognition site and is potentially relevant to a hypothesis that this rRNA region undergoes an ordered series of conformational changes during the ribosome cycle.

Base Sequence↗
PubMed1989

S4-16 S ribosomal RNA complex. Binding constant measurements and specific recognition of a 460-nucleotide region.

The region of the Escherichia coli 16 S ribosomal RNA recognized by the ribosomal protein S4 has been defined by assaying a set of 13 16 S rRNA fragments for S4 binding. The fragments were prepared by transcription in vitro, and binding constants were measured in three ways: retention of labeled RNA fragments on nitrocellulose filters by S4; co-sedimentation of labeled S4 with RNA fragments in sucrose gradients; and the distribution of labeled S4 between two RNAs of different sizes in a sucrose gradient. All three methods gave similar relative binding strengths for a variety of 16 S rRNA and non-specific (23 S rRNA) sequences, with the exception of two of the largest 16 S rRNA fragments; these gave smaller association constants in the filter retention assay than in the other methods. We found that specific complexes of S4 with these larger RNAs do not bind well to filters, leaving non-specific complexes to dominate the assay. Specific complexes with RNAs less than or equal to 891 nucleotides were retained efficiently by S4 on filters, and gave reliable binding constants. All 16 S rRNA fragments containing nucleotides 39 to 500 bound S4 with the same affinity as intact 16 S rRNA, while all fragments with endpoints within 39 to 500 bound at least tenfold more weakly. This sequence must be able to fold independently of the rest of the rRNA. Comparison of this minimal 462-nucleotide S4 binding site with S4 footprinting results suggests that S4 binding might alter the conformations of RNA neighboring the 39 to 500 region in the intact 16 S rRNA. Specific S4-rRNA binding is not sensitive to KCl concentration, but a more normal salt dependence is seen in K2SO4 (delta logK/delta log[K+] approximately -3.3). This duplicates the behavior of the specific S4-alpha mRNA translational repression complex, arguing that S4 recognizes both the mRNA and rRNA substrates by the same mechanism. Mg2+ is not required to form the specific rRNA complex, at least under conditions which stabilize RNA structure (0.35 M-KCl, 5 degrees C).

Binding Sites↗
PubMed1989

Unusual mRNA pseudoknot structure is recognized by a protein translational repressor.

Translation of ribosomal proteins in the alpha operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression.

Base Composition↗
PubMed1989

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment.

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.

Base Sequence↗
PubMed1989

How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins.

Some ribosomal proteins which bind specifically to ribosomal RNA also act as translational repressors and recognize their encoding messenger RNAs. The messenger- and ribosomal-RNA binding sites for four of these proteins are now well defined, and striking similarities in primary and secondary structure are apparent in most cases. These 'consensus' structures are useful clues to the features proteins use to recognize specific RNAs.

Base Sequence↗
PubMed1987

S4-alpha mRNA translation repression complex. I. Thermodynamics of formation.

Expression of the four ribosomal proteins from the Escherichia coli alpha operon (S4, S11, S13, and L17) is regulated at the level of translation by the binding of S4 to the alpha mRNA. Using a filter binding assay and alpha mRNA sequences prepared by in-vitro transcription, previous work located the S4 target site within the approximately 100-base leader sequence. We have extended this work to include fragments of the alpha leader with six different 5' end points and four different 3' end points. A core region between bases 23 and 69 (numbering from the first nucleotide of the E. coli transcript) binds S4 with an affinity of approximately 2 microM-1. Regions of weak interactions are located in the 22 nucleotides 5' and the 70 nucleotides 3' to this core; they increase the S4 affinity to approximately 13 microM-1. Studies of S4-alpha mRNA binding under different conditions have revealed the following. (1) Specific and non-specific binding show the same dependence on K+ concentration, with delta log+ K/delta log [K+] approximately 4 in most potassium salts. With KCl and KBr, much weaker salt dependence of specific complex formation is observed suggesting that the protein responds to the correct RNA substrate by binding halide anions. (2) Increasing the MgCl2 concentration between 1 and 4 mM enhances binding by a factor of 4, with no further effects up to 20 mM. About five Mg2+ are taken up by the complex with an average binding constant of approximately 600 M-1 each. Renaturation of the RNA in the presence of MgCl2 is also required to obtain full binding. These effects are seen only with alpha mRNA extending beyond the initiation codon; S4 binding to the alpha leader sequence itself is insensitive to Mg2+. (3) The association kinetics are fast and probably diffusion controlled. (4) Formation of the complex is entirely entropy driven.

Base Sequence↗
PubMed1987

S4-alpha mRNA translation regulation complex. II. Secondary structures of the RNA regulatory site in the presence and absence of S4.

The secondary structure of the Escherichia coli alpha mRNA leader sequence has been determined using nucleases specific for single- or double-stranded RNA. Three different length alpha RNA fragments were studied at 0 degrees C and 37 degrees C. A very stable eight base-pair helix forms upstream from the ribosome initiation site, defining a 29 base loop. There is evidence for base-pairing between nucleotides within this loop and for a "pseudo-knot" interaction of some loop bases with nucleotides just 3' to the initiation codon, forming a region of complex structure. A weak helix also pairs sequences near the 5' terminus of the alpha mRNA with bases near the Shine-Dalgarno sequence. Affinity constants for the translational repressor S4 binding different length alpha mRNA fragments indicate that most of the S4 recognition features must be contained within the main helix and hairpin regions. Binding of S4 to the alpha mRNA alters the structure of the 29 base hairpin region, and probably melts the weak pairing between the 5' and 3' termini of the leader. The pseudo-knot structure and the conformational changes associated with it provide a link between the structures of the S4 binding site and the ribosome binding site. The alpha mRNA may therefore play an active role in mediating translational repression.

Autoradiography↗
PubMed1987

Single base bulges in small RNA hairpins enhance ethidium binding and promote an allosteric transition.

A set of four RNA hairpin helices has been prepared by in vitro transcription with T7 RNA polymerase. The hairpins all contain the same nine base pair helix, but with an extra A, C, or U residue forming a bulge at one position; the fourth hairpin is a perfect helix with no bulge. The helix with a bulged A duplicates six base pairs of a helix in the 16S rRNA known to have an unusually high affinity for ethidium bromide [J. M. Kean, S. A. White, and D. E. Draper, Biochemistry 24, 5062 (1985)]. Binding and chemical cleavage studies with ethidium or the reagent methidiumpropylEDTA-Fe(II) [MPE-Fe(II)] showed that the sequence CpG is a preferred intercalation site whether or not a bulge is present; all three bulged bases enhance intercalation at the CpG sequence by an order of magnitude; and intercalation in a bulged helix results in a concerted conformational change involving the entire helix backbone, again dependent on the presence of a bulge but independent of the particular base. These results suggest that an extra sugar-phosphate residue in an RNA helix backbone has a dramatic effect on the ability of the RNA to adopt new conformations. This effect could be an important reason for the conservation of bulges at certain positions in ribosomal and other RNAs.

Allosteric Site↗
PubMed1986

On the recognition of helical RNA by cobra venom V1 nuclease.

The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the utility of this enzyme as an RNA structure probe, we have investigated its properties and substrate specificity, using assays for polynucleotide hydrolysis based on fluorescent polynucleotide derivatives. Enzymatic activity requires both Na+ and Mg2+, with optima at 100 and 0.3 mM, respectively. From the sharp decrease in enzyme activity above 100 mM Na+ we estimate that 3-4 ionic interactions between the protein and polynucleotide phosphates take place. Analysis of products remaining after extensive V1 digestion also shows that the minimum size substrate is 4-6 nucleotides long. Helical RNAs and DNAs have Michaelis constants a factor of 3-10 times lower than most single-stranded RNAs. However, poly(epsilon A) has a Michaelis constant equal to the best synthetic double helices tested and is hydrolyzed at a rate comparable to helical RNA. The major V1 cutting sites in yeast tRNAPhe have Michaelis constants lower than any synthetic polymers. These data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix. A few single-stranded V1 cleavage sites are known in tRNA and rRNA, and their structures are consistent with the suggested V1 recognition site.

Base Sequence↗
PubMed1985

Specific interaction between ribosomal protein S4 and the alpha operon messenger RNA.

The Escherichia coli ribosomal protein S4 is known to repress translation of its own gene and several other ribosomal protein (r-protein) genes in the alpha operon as part of a general mechanism coordinating the levels of rRNA and r-protein synthesis. Using a filter binding assay and RNA transcripts prepared in vitro, we have detected and quantitated specific interactions between S4 and alpha mRNA fragments. The main results are the following: Only the alpha mRNA leader is required for specific recognition, with a small fraction of the binding free energy derived from sequences at the ribosome initiation site. 16S rRNA and alpha mRNA compete for binding to S4 with about the same affinity (approximately equal to 2 X 10(7) M-1), suggesting that S4 utilizes the same recognition features in each RNA. Nonspecific binding of S4 to tRNA or other mRNA sequences is strongly salt dependent, while the specific S4-alpha mRNA affinity is nearly independent of salt. At physiological salt concentrations the nonspecific S4-RNA affinity (10(5)-10(6) M-1) is large enough to strongly buffer the free S4 concentration in vivo.

Escherichia coli↗