Hydroxyl Radical Footprinting Experiments Research Articles

Shape changes and cooperativity in the folding of the central domain of the 16S ribosomal RNA

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0 and 30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

Hydroxyl-radical footprinting combined with molecular modeling identifies unique features of DNA conformation and nucleosome positioning.

Nucleosomes are the most abundant protein–DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein–DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197bp nucleosome bearing symmetric 25bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.

Time-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS.

The virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NSctd), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The ‘fast’, ‘intermediate’ and ‘slow’ H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all ‘fast’ protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps.

Activation of Archaeal Transcription Mediated by Recruitment of Transcription Factor B

Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested.

Protein-DNA interactions define the mechanistic aspects of circle formation and insertion reactions in IS2 transposition

BackgroundTransposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.ResultsIn IS2, Step I reactions are physically and chemically asymmetric; the left imperfect, inverted repeat (IRL), the exclusive recipient end, lacks donor function. In SC I, different protection patterns of the cleavage domains (CDs) of the right imperfect inverted repeat (IRR; extensive in cis) and IRL (selective in trans) at the single active cognate IRR catalytic center (CC) are related to their donor and recipient functions. In SC II, extensive binding of the IRL CD in trans and of the abutted IRR CD in cis at this CC represents the first phase of the complex. An MCJ substrate precleaved at the 3' end of IRR revealed a temporary transition state with the IRL CD disengaged from the protein. We propose that in SC II, sequential 3' cleavages at the bound abutted CDs trigger a conformational change, allowing the IRL CD to complex to its cognate CC, producing the second phase. Corroborating data from enhanced residues and curvature propensity plots suggest that CD to CD interactions in SC I and SC II require IRL to assume a bent structure, to facilitate binding in trans.ConclusionsDifferent transpososomes are assembled in each step of the IS2 transposition pathway. Recipient versus donor end functions of the IRL CD in SC I and SC II and the conformational change in SC II that produces the phase needed for symmetrical IRL and IRR donor attacks on target DNA highlight the differences.

Oligomeric Structure of the Chemokine CCL5/RANTES from NMR, MS, and SAXS Data

CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration invitro, CCL5's propensity for aggregation is essential for migration invivo, Tcell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration invivo.

Measuring the Dimensions of a Compact Kinetic Intermediate in the Folding Pathway of the GlmS Ribozyme

Using complementary time-resolved biochemical and x-ray probes of RNA structure in solution, we investigate the cation-induced folding of the glmS ribozyme, a metabolite-sensing RNA switch that regulates gene expression in bacteria. Hydroxyl radical footprinting experiments have shown a concerted folding transition within the first 10 seconds after adding magnesium. From small angle x-ray scattering (SAXS) experiments performed under similar conditions, we find that native tertiary contact formation is preceded by the collapse of the molecule to a relatively compact intermediate. The subsequent compaction observed by SAXS correlates temporally with changes in hydroxyl radical protection. We propose a structural model for the intermediate and possible implications for the role of secondary structure and electrostatics in the folding process of this ribozyme.

Aliphatic Peptidyl Hydroperoxides as a Source of Secondary Oxidation in Hydroxyl Radical Protein Footprinting

Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.

Identifying Energy Barriers in RNA Folding Through Kinetic Model Enumeration

Large RNA molecules are known to fold through multiple parallel pathways to achieve their functional conformation. These pathways include stable intermediate structures that we identify using our KinFold algorithm (http://simtk.org/home/kinfold) for the analysis of time-resolved hydroxyl radical footprinting experiments. KinFold enumerates kinetic model topologies to determine the structure, lifetimes, and abundance of intermediates along the folding pathways of RNA. We are therefore able to identify the best fitting kinetic model for each folding reaction studied. We used this approach to study the folding of the L-21 T. thermophila group I intron, a catalytic ribozyme, at 6 temperatures ranging from 25° to 51° C. Our analysis of this data reveals that the rates for a majority of the transitions (between intermediate, folded and unfolded states) obey the Arrhenius equation, allowing us to estimate the relative energies of activation for inter conversion between the states of an RNA. Furthermore, we are able to estimate the lifetimes of the different intermediate structures. These results reveal a simple landscape, where folding of the peripheral elements of the RNA create significant barriers to folding. Our approach is unique in that it allows us to simultaneously estimate the activation energies for all transitions in an RNA folding reaction.

Footprinting protein–DNA complexes using the hydroxyl radical

Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA-protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.

Interaction of the PHD-Finger Homeodomain Protein HAT3.1 from Arabidopsis thaliana with DNA. Specific DNA Binding by a Homeodomain with Histidine at Position 51

HAT3.1 is a member of the PHD-finger homeodomain protein family. The HAT3.1 homeodomain is highly divergent in sequence even at positions that are almost invariable among homeodomains. In this work, we have applied the random oligonucleotide selection technique to investigate if the HAT3.1 homeodomain is able to recognize specific DNA sequences. Analysis of the selected molecules followed by hydroxyl radical footprinting experiments and yeast one-hybrid assays indicated that HAT3.1 shows a preference for the sequence T(A/G)(A/C)ACCA, different from those bound by other homeodomains. Binding was dependent on homeodomain residues located at positions 47, 50, 51, and 54, the same positions that usually participate in DNA binding in most homeodomains. The study of the interaction of mutants at these positions with DNA carrying nucleotide changes at specific sites suggested that H51 and K50 most likely interact with nucleotides 2 to 4 and 5 to 6, respectively, while W54 would establish contacts with position 4. The presence of H51 and W54 represents an innovation among homeodomain structures. The fact that the HAT3.1 homeodomain is able to interact with specific DNA sequences is evidence of the inherent plasticity of the homeodomain as a DNA binding unit.

The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms.

The Tn10 transpososome has symmetrical components on either side: there are two transposon ends each of which has binding sites for a monomer of transposase and an IHF heterodimer. The DNA bending activity of IHF stimulates assembly of an intermediate with tightly folded transposon ends in which transposase has additional 'subterminal' DNA contacts, located distal to the IHF site. These subterminal contacts are required to activate later steps in the reaction. Quantitative hydroxyl radical footprinting and gel retardation unfolding experiments show that the transpososome is fundamentally asymmetric, despite having identical components on either side. Major differences between the transposon ends define alpha and beta sides of the complex. IHF can dissociate from the transposon arm on the beta side of the complex in the absence of metal ion. However, IHF is locked onto the alpha side of the complex, probably by the subterminal transposase contacts, until released by a metal ion-dependent conformational change. Later in the reaction, IHF inhibits target interactions. Using a very short transposon arm, target interactions are demonstrated at a saturating IHF concentration. This suggests that inhibition of target interactions is due to steric hindrance of the target binding site by a single IHF-folded transposon arm.

Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR.

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.

The Yeast Transcription Factor Mac1 Binds to DNA in a Modular Fashion

Mac1 is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae. Under conditions of high copper concentration, Mac1 represses transcription of genes coding for copper transport proteins. Mac1 binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3'. Mac1 contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of A/T-rich sequences in DNA. We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter. We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.

Substrate recognition by Escherichia coli MutY using substrate analogs

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.

Interaction of MutS Protein with the Major and Minor Grooves of a Heteroduplex DNA

Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of Taq MutS protein to a heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24-28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish that Taq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1, 10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA.

Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium

CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.

Structure-Function Relationship of the Lrp-binding Region Upstream of lysU in Escherichia coli

In Escherichia coli, one of the two genes encoding lysyl-tRNA synthetase, lysU, belongs to the regulon controlled by the leucine-responsive protein (Lrp). To map the site of Lrp action, mutants escaping regulation in rich medium were generated through random mutagenesis of the lysU promoter region. The mutations showed parallel effects on the strength of Lrp-DNA association, as measured in vitro by gel retardation experiments, and on the degree of repression of lysU expression by Lrp in vivo . In addition, DNase I and hydroxyl radical footprinting experiments indicated that several Lrp molecules bind to a DNA region of over 110 bp in a highly cooperative manner. This region, which encompasses the -35 box of the lysU promoter, was the target of all the mutations affecting the strength of the Lrp-DNA association. These mutations are frequently located in short A + T-rich runs distributed along the Lrp binding region with a periodicity of one helix turn. Because we could find such a regular alternance of A + T runs upstream of several other Lrp-regulated genes, we suggest that this pattern is one feature indicative of the binding of Lrp.

Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3' untranslated region of alfalfa mosaic virus RNA 4

The specific binding of alfalfa mosaic virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus coat protein and ilavirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements.