Why Do Rna Read Major Groove and Not Minor Groove on Dna
Nature. Writer manuscript; available in PMC 2010 Apr 29.
Published in final edited grade as:
PMCID: PMC2793086
NIHMSID: NIHMS143859
The role of DNA shape in poly peptide-Deoxyribonucleic acid recognition
Remo Rohs
1Howard Hughes Medical Establish, Centre for Computational Biology and Bioinformatics, Department of Biochemistry and Molecular Biophysics, Columbia University, 1130 St. Nicholas Avenue, New York, NY 10032, United states
Sean Chiliad. W
aneHoward Hughes Medical Institute, Heart for Computational Biology and Bioinformatics, Section of Biochemistry and Molecular Biophysics, Columbia University, 1130 St. Nicholas Avenue, New York, NY 10032, The states
Alona Sosinsky
1Howard Hughes Medical Institute, Center for Computational Biology and Bioinformatics, Department of Biochemistry and Molecular Biophysics, Columbia University, 1130 St. Nicholas Avenue, New York, NY 10032, USA
Peng Liu
1Howard Hughes Medical Plant, Heart for Computational Biological science and Bioinformatics, Section of Biochemistry and Molecular Biophysics, Columbia Academy, 1130 St. Nicholas Avenue, New York, NY 10032, U.s.
Richard S. Mann
2Department of Biochemistry and Molecular Biophysics, Columbia Academy, 701 West 168th Street, HHSC 1104, New York, NY 10032, U.s.a.
Barry Honig
oneHoward Hughes Medical Institute, Heart for Computational Biology and Bioinformatics, Section of Biochemistry and Molecular Biophysics, Columbia Academy, 1130 St. Nicholas Avenue, New York, NY 10032, U.s.
Abstruse
The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanisms: 1 that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. Past comprehensively analyzing the iii dimensional structures of protein-Dna complexes, we show that the binding of arginines to narrow small grooves is a widely used fashion for protein-Deoxyribonucleic acid recognition. This readout machinery exploits the miracle that narrow minor grooves strongly enhance the negative electrostatic potential of the Dna. The nucleosome core particle offers a striking case of this effect. Minor groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings advise that the ability to detect local variations in Dna shape and electrostatic potential is a full general mechanism that enables proteins to utilise information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA bounden specificity.
The ability of proteins to recognize specific DNA sequences is a hallmark of biological regulatory processes. The determination of the iii-dimensional structures of numerous protein-DNA complexes has provided a detailed picture of binding, revealing a structurally diverse set of protein families that exploit a wide repertoire of interactions to recognize the double-helixi. Nucleotide sequence-specific interactions frequently involve the formation of hydrogen bonds betwixt amino acid side chains and hydrogen bond donors and acceptors of individual base of operations pairs. It has long been recognized that every base pair has a unique hydrogen bonding signature in the major groove but that this is not the case in the modest groove2. Thus, the expectation has been that the recognition of specific DNA sequences would have place primarily in the major groove through the formation of a series of amino acid- and base-specific hydrogen bonds1. This "direct readout" machinery is consistent with observations derived from 3-dimensional structures of poly peptide-DNA complexes simply it is far from the entire story.
In many complexes, the Deoxyribonucleic acid assumes conformations that deviate from the structure of an platonic B-grade double helixthree – 5, sometimes bending in such a way to optimize the protein-DNA interfacehalf dozen and in some cases undergoing significant conformational changes as in the opening of the modest groove in the complex formed between TBP and the TATA box7 , 8. The term "indirect readout" was coinedix to describe such recognition mechanisms that depend on the propensity of a given sequence to presume a conformation that facilitates its bounden to a particular poly peptide. The bases involved in such mechanisms need not exist in contact with the protein and, for example, can exist establish in linker sequences that connect two half-sites that themselves are bound by private poly peptide subunits10 , 11.
We recently described an example of a novel readout machinery, the recognition of local sequence-dependent minor groove shape12, that is distinct from previously described indirect readout mechanisms. In this case, the sequence-dependence of minor groove width and respective variations in electrostatic potential are used by the Hox protein Sexual practice combs reduced (Scr) to distinguish small differences in nucleotide sequence12. Here we report that this mechanism is a widely used manner of protein-Deoxyribonucleic acid recognition that involves the creation of specific bounden sites for positively charged amino-acids, primarily arginine, within the minor groove. Minor groove narrowing is found to be correlated with A-tracts13 , 14, usually defined every bit stretches of four or more than Equally or Ts that do not contain the flexible TpA pace15, but extended here to include as few as three base pairs (see beneath). Our results offering fundamentally new insights into the structural and energetic origins of protein-Dna binding specificity and thus take of import implications for the prediction of transcription cistron binding sites in genomes.
Arginine is enriched in narrow minor grooves
Figure 1a reports the percentage of small groove contacts associated with each amino acid, classified according to the width of the pocket-sized groove. Arginine constitutes 28% of all amino acid residues that contact the minor groove and is significantly enriched in narrow modest grooves, divers hither past a groove width of <5.0 Å (compared to v.8 Å in ideal B-DNA). Remarkably, 60% of the residues in narrow pocket-size grooves are arginines as compared to 22% in small-scale grooves that are defined as not narrow – i.eastward. width ≥v.0 Å. A smaller enrichment is also observed for lysines only the overall population of lysines within the pocket-size groove is much less than for arginine.
Amino acid frequencies in minor grooves
(a) Histograms for each amino acid illustrate the frequency with which they are observed in any minor groove (dark-green), in minor grooves with a width of ≥v.0 Å (blue), and in narrow minor grooves of <5.0 Å width (scarlet). (b) Frequency of AT (Due west) and GC (S) base pairs in sequences of 229 sites contacted by arginines in narrow minor grooves. The central base pair (boxed) is contacted by arginine. Frequencies are symmetrized past using both complementary strands.
Binding to the minor groove is a characteristic of many, but not all, protein superfamilies and a significant subset of these contact a narrow minor groove (Table 1). Moreover, if the minor groove is contacted, arginines are likely to be involved, and the likelihood that an arginine will be present becomes even greater for narrow small-scale grooves (Supplementary Table ane).
Table 1
Protein superfamilies with minor groove contacts.
| SRF-like |
| IHF-like Dna-bounden proteins |
| Histone-fold |
| Deoxyribonucleic acid breaking-rejoining enzymes |
| Zn2/Cys6 DNA-binding domain |
| Homeodomain-similar |
| p53-like transcription factors |
| lambda repressor-similar Dna-binding domains |
| Winged helix Dna-bounden domain |
| Leucine zipper domain |
| C-final effector domain of the bipartite response regulators |
| Brake endonuclease-similar |
| Glucocorticoid receptor-like (Dna-bounden domain) |
| DNA repair protein MutS, domain I |
| Origin of replication-binding domain, RBD-like |
| DNA/RNA polymerases |
| Eukaryotic Deoxyribonucleic acid topoisomerase I, Northward-terminal Deoxyribonucleic acid-binding fragment |
| Ribonuclease H-similar |
| TATA-box binding protein-like |
Listed are SCOP superfamilies47 that have an arginine-modest groove contact inside a distance of <half-dozen.0 Å from the base of operations. Superfamilies that use arginine to contact a narrow modest groove (<5.0 Å) have grey shading; those that utilise arginine to contact a not-narrow minor groove (≥5.0 Å) are unshaded. Only superfamilies with a minimum of ten protein chains in PDB structures spring to DNA at to the lowest degree 1 helical plow long are included. The percentages of chains with modest groove contacts vary considerably among SCOP superfamilies and are provided in Supplementary Table 1.
Figure 1b compiles the Dna sequence preferences for protein-Deoxyribonucleic acid complexes in which an arginine contacts a narrow minor groove. The effigy shows that the base of operations pair that has the shortest contact distance with the arginine guanidinium grouping has a probability of 78% of beingness an AT and 22% of being a GC. Neighboring base of operations pairs in both the v' and 3' directions surrounding the closest contacting base pair also have a strong tendency to be AT. Taken together, these data demonstrate that arginines tend to demark narrow minor grooves in AT-rich Deoxyribonucleic acid.
AT-rich sequences tend to narrow minor grooves
Nosotros calculated small-scale groove widths for all tetranucleotides contained in PDB structures for both complimentary Deoxyribonucleic acid (Effigy 2a) and DNA in complexes with proteins (Effigy 2b). At that place is a big spread of values due in part to end effects and to the effects of crystal packing merely some trends are withal axiomatic. For case, for free Dna structures near of the tetranucleotides with narrow minor grooves (width <5.0 Å) are AT-rich (Figure 2a and Supplementary Table 2a). Similar behavior is observed in protein- DNA complexes (Figure 2b and Supplementary Table 2b). In dissimilarity, tetranucleotides with wide pocket-sized grooves have a strong tendency to be GC-rich.
Distribution of tetranucleotide sequences according to average minor groove width
Tetranucleotides from structures with a minimum length of one helical turn for which minor groove width tin can be defined are ordered by average pocket-size groove width (red). The widths of all tetranucleotides are shown (black) and the sequence, boilerplate width, and occurrence in our dataset are given in Supplementary Tabular array 2. (a) The 59 unique tetranucleotides from complimentary DNA structures. (b) The set of all 136 unique tetranucleotides derived from protein-Deoxyribonucleic acid complexes.
The correlation between AT content and groove width is not unexpected given the fact that A-tracts are known to produce narrow minor grooves. All the same, TpA steps take a tendency to widen the minor groovefifteen, and then it was of interest to decide whether the distinct properties of A-tracts and TpA steps are reflected in our tetranucleotide data set. We find that 67% of tetranucleotides composed only of AT base pairs accept a narrow modest groove but that this number increases to 82% if we exclude TpA steps so as to consider only A-tracts. Even A-tracts of length three accept a strong tendency to narrow the minor groove. Forty three percent of the tetranucleotides with a pocket-size groove width of <5.0 Å take an A-tract of length three, a percentage that decreases to eleven% of tetranucleotides with canonical minor groove widths (between 5.0 and 7.0 Å) and to 4% of tetranucleotides with small grooves wider than 7.0 Å (Supplementary Figure one). Additionally, compared to other AT-rich sequences, A-tracts are specifically enriched in DNAs with narrow minor grooves (Supplementary Figure 1). Thus, although A-tracts are usually thought of as requiring four or more base of operations pairs, in part because a minimum of four is required to rigidify the DNAfourteen, this analysis shows that A-tracts as short every bit length three are positively correlated with narrow pocket-size grooves.
Arginines recognize enhanced electrostatic potentials
Figure 3 and Supplementary Figure two plot minor groove width and electrostatic potential vs. bounden site sequence for several complexes whose binding interface includes an arginine inserted into the minor groove. The correlation of width and potential besides as the tendency of arginines to be located close to minima in width and potential is evident. Below nosotros highlight a few specific examples of how arginine-minor groove interactions are used in DNA recognition.
Specific examples of pocket-sized groove shape recognition by arginines
Dna shapes of the binding sites of (a) Ubx-Exd 1b8i16, (b) MATa1/MATα2 1akh17, and (c) Oct-i/PORE 1hf018, (d) the MogR repressor 3fdqxix, (e) the Tc3 transposase 1u7822, and (f) the phage 434 repressor 2or123 are shown in GRASP surface representations31 , 50 with convex surfaces color-coded in green and concave surfaces in grey/black. Plots of minor groove width (blue) and electrostatic potential in the eye of the minor groove (crimson) are shown below. Arginine contacts (defined by the closest distance betwixt the guanidinium groups and the bases) are indicated. A-tract sequences are highlighted by a solid red line, the TATA box in (e) by a dashed line.
Figure 3a represents the ternary complex of the Hox poly peptide Ultrabithorax (Ubx) and its cofactor Extradenticle (Exd) bound to DNA16. In this complex, Arg5 of Ubx, which is a conserved residuum across all homeodomains, inserts into a narrow region formed by a four base of operations pair A-tract. Figures 3b provides an case of a long and very narrow A-tract that binds α2-Arg7 from the MATa1/MATα2 complex with DNA17. In dissimilarity, α2-Arg4 inserts into a shallower region at one end of the A-tract where in that location are local minima in width and potential that are smaller than at the Arg7 site in the eye of the A-tract. The two POU-domains of the Oct-i/PORE circuitous bind to two A-tracts (Figure 3c) where the minima are positioned in such a fashion to provide binding sites for 4 arginines, two from each POU domaineighteen.
The location of these A-tracts with respect to other nucleotide sequence features tin can exist used to generate specificity, as previously discussed for the Hox poly peptide Scr12. In the case of Scr binding, the position of a TpA pace within an AT-rich region plays a disquisitional role in binding specificity. A similar strategy is used by the MogR transcription cistron where two long A-tracts separated by a TpA step produce two arginine bounden sites19 (Effigy 3d). The unique shape recognized by these 2 arginines is likely to contribute to the position of the MogR bounden site along the DNA sequence. The overall tendency of TpA steps to widen the pocket-sized groove is nearly apparent when they are positioned between two A-tracts (as in Scr12 and MogR19) where the TpA step acts equally a `hinge' between more rigid elements15 , twenty. In other contexts, due to their flexibility, TpA steps can also be accommodated in narrow small-scale grooves21. An example is provided by the bipartite DNA-binding domain of Tc3 transposase where the arginines bind to a narrow region containing a TATA box22 that displays enhanced negative electrostatic potential (Figure 3e).
Although less frequent, arginines also bind narrow grooves associated with non-A- tract sequences. Effigy 3f summarizes features of the binding of the 434 repressor to its operator23 which contains seven base pairs that are all AT with the exception of a central CG. (The guanine amino group tends to widen narrow grooves simply a single GC base pair tin be accommodated with merely trivial disruption.)
Arginine-small groove interactions in the nucleosome
Figure 4a plots minor groove width and electrostatic potential forth the Deoxyribonucleic acid sequence of the nucleosome cadre particle containing recombinant histones and a 147 base of operations pair DNA fragment (PDB code 1kx5)24. There are 14 minima in minor groove width respective to regions where the Deoxyribonucleic acid bends so equally to wrap around the histone cadre. Every bit in a higher place, there is a striking correlation between width and potential. The variation in width between the narrowest and widest regions is about 5 Å and the difference between the maxima and minima in electrostatic potential is nigh 6 kT/east (Figure 4a). As a event, in that location should be a potent driving force for basic amino acids to bind to narrow regions and indeed arginines are establish in nine of the fourteen minima. These arginines are shown in Figure 4b where the nucleosomal DNA has been color coded past minor groove width. (Although all 14 narrow small groove regions are contacted by arginines24 only nine of these satisfy our criteria of <6.0 Å between arginine atoms and base atoms in the groove). A like repeating pattern of narrow minor grooves that are contacted by arginines is seen in all 35 available nucleosome crystal structures (Supplementary Figure 3a,b).
Modest groove shape recognition in the nucleosome
(a) Correlation of minor groove width of the nucleosome core particle (PDB code 1kx5)24 (blue) and electrostatic potential (cherry-red). Arginine contacts (divers by the closest altitude between the guanidinium groups and the bases) are indicated. A-tract sequences are highlighted past solid red lines. (b) Schematic representation of the Deoxyribonucleic acid backbone in the nucleosome color-coded by minor groove width (red ≤4.0 Å, pinkish >4.0 Å and ≤5.0 Å, light blueish >v.0 Å and ≤vi.0 Å, dark blue >half dozen.0 Å), including all arginines that contact the minor groove. (c) The distribution of A-tracts of length 3 base pairs or longer in 23,076 yeast nucleosome-bound Deoxyribonucleic acid sequences29. (d) Histogram of the occurrence of A-tracts of length three or longer in the same dataset29.
Considering short A-tracts narrow the modest groove and facilitate the bending of DNA, we would expect to meet a periodicity of A-tracts in DNA sequences bound by nucleosomes in vivo. Previous analyses have focused on dinucleotide statistics25 , 26 although it has been known for some time that there is a periodic pattern of AAA and AAT trinucleotides in nucleosome core Dna27 , 28. An assay of DNA sequences spring in vivo by yeast nucleosomes29 reveals a articulate periodicity for A-tracts of at least length three (denoted 3+, Figure 4c). Moreover, nucleosomal DNAs contain, on average, 10.0 A-tracts of length 3+ (Figure 4d). Periodicity is likewise detected for A-tracts of length four+ and even v+, although the number per nucleosome decreases to 4.1 and one.vi, respectively (Supplementary Effigy 3). Thus, even though long A-tracts tend to exist excluded from the nucleosomexxx, A-tracts of ≤ five base pairs, when present, are used to facilitate bending of the Dna around the histone cadre.
To evaluate the outcome of TpA steps, we compared the periodicities of A-tracts of length three to those of other trinucleotides equanimous just of AT base pairs. Trinucleotides that contain TpA steps showroom a much weaker periodic signal than A-tracts of length three, which exclude the TpA step (Supplementary Figure 4). Together, this analysis suggests that many of the sequence periodicities in nucleosomal Dna reflect the presence of short A-tracts that pb to narrow regions in the minor groove that in plough are recognized past a complementary set of arginines nowadays on the surface of the nucleosome core particle.
Effects of groove width on electrostatic potential
The remarkable correlation between minor groove width and electrostatic potential (Figures 3 and 4) is due primarily to the properties of the Poisson-Boltzmann (PB) equation that have been extensively discussed in the literature31. Biological macromolecules are less polarizable than the aqueous solvent and, in the language of classical physics, tin can be thought of as a depression dielectric region embedded in a high dielectric solvent. Solutions of the Pb equation for DNA showed that lines of electrostatic potential due to backbone phosphates follow the shape of the Deoxyribonucleic acid and are the almost negative inside the grooves32. This upshot is due to electrostatic focusing, beginning observed for the protein superoxide dismutase31, where the narrow agile site focuses electric field lines away from the protein and into the high dielectric solvent. The same concrete miracle produces enhanced potentials in grooves, accounting for the stiff correlation described higher up.
In order to constitute the source of the consequence in quantitative terms, we calculated the potentials for the MogR binding site19 when the dielectric abiding is set to 80 both within the macromolecule and in the solvent (Figure v, dashed line) and for the case where the 2 dielectric constants are unlike (Figure 5, solid line). Strikingly, a significant enhancement of electrostatic potentials is simply observed when the dielectric constant of the macromolecule and solvent are different, reflecting the focusing of electric field lines described qualitatively above. The small outcome seen when the dielectric constant is the same results from the phosphates being closer to the center of the groove when it is narrow (see Supplementary Effigy five for a breakdown of the contributions to the net electrostatic potential). Both sets of calculations were carried out at physiological salt concentrations. Although ionic force has every bit stiff effect on the absolute values of the potentials, the effect remains qualitatively the same (Supplementary Figure six).
The biophysical origins of the negative potential of narrow small-scale grooves
Electrostatic potential in the pocket-size groove of the MogR binding site (PDB code 3fdq)19, calculated in the presence of a dielectric purlieus (ε=ii in solute and ε=lxxx in solvent – solid line) and in the absenteeism of a boundary (ε= 80 in both solute and solvent – dashed line).
Why are arginines preferred over lysines?
Information technology is somewhat surprising that there is such a significant population of arginines in the minor groove, and a big enrichment when the groove is narrow, whereas the effects for lysines are more modest (Figure 1a). Arginines have been known for some time to exist enriched relative to lysines in protein-protein33 and protein-Dna interfaces34 and the difference has generally been attributed to the ability of the guanidinium group to engage in more hydrogen bonds than the amino group of lysine35. To evaluate this idea we adamant the number of hydrogen bonds formed by all the arginines and lysines in our data set that penetrate the minor groove. Surprisingly, on average, less than one hydrogen bail is formed by either amino acid side chain to Dna (0.9 for arginine and 0.6 for lysine), and the standard deviations are such that this difference is insignificant (Supplementary Table 3).
An alternate explanation derives from the difference in the size of the cationic moieties of the two residues. According to the classical Built-in model the solvation gratis energies of ions are proportional to the inverse of their radii31, suggesting that it is energetically less costly to remove a charged guanidinium group from water than it is to remove the smaller amino group of a lysine. To test this quantitatively, we calculated the change in gratis energy in transferring arginine and lysine from water to a medium of dielectric abiding ii (see Methods for details). The difference in the transfer free energies betwixt the two residues ranges from ii.3 to half-dozen.7 kcal/mole, depending on the force field that was used, with lysine consistently having the higher value (Supplementary Table four). These results suggest that the college prevalence of arginines compared to lysines in pocket-sized grooves is due, at least in office, to the greater energetic cost of removing a charged lysine from water than to remove a charged arginine.
Final remarks
We take shown that in that location is a dramatic enrichment of arginines in narrow regions of the Deoxyribonucleic acid pocket-size groove that provides the basis for a novel DNA recognition mechanism that is used by many families of DNA-binding proteins. A readout mechanism based on groove width requires a connectedness betwixt sequence and shape. This connection appears to be provided in part by A-tracts, which accept a stiff tendency to narrow the groove, producing bounden sites for arginines that, when spaced accordingly on the protein surface, offer a complementary set up of positive charges that can recognize local variations in shape. Arginines often insert into the minor groove as part of brusk sequence motifs (eastward.g. RQR in the Hox protein Scr12, RKKR in POU homeodomainsxviii, RPR in Engrailed36, RGHR in MATa1/MATα217, RRGR in the nuclear orphan receptor37 and RGGR in the homo orphan receptor38), thus offering a diverseness of presentation modes that can contribute to the specificity of Dna shape recognition.
The tendency of A-tracts to narrow the minor groove is due primarily to their power to assume conformations, through propeller twisting, that atomic number 82 to the formation of inter- base pair hydrogen bonds in the major groove15. This network is disrupted by TpA steps as strikingly seen in the MogR bounden site19. GC base of operations pairs besides take a trend to widen the minor groovexiv. The combination of these and other factors, such equally effects induced by flanking bases that are non directly located within the bounden site39, can produce a complex small-scale groove landscape that offers numerous possibilities for specific interactions with proteins. Indeed, minor groove geometry is no doubt the result of the interplay of intrinsic and protein-induced structural furnishings.
The physical mechanisms described here are dramatically axiomatic in the nucleosome. The energetic cost of narrowing and angle the Dna in regions where the backbone faces inward volition be reduced by the presence of curt A-tracts that take an intrinsic propensity to assume such conformations and hence to bend the DNA28. In addition, the penetration of arginines into the minor groove at sites where the DNA bends and the groove is narrow21 , 40 provides a significant stabilizing interaction
The variations in Dna shape observed in poly peptide-DNA complexes often reflect conformational preferences of gratis DNAiv , 10 , 41. Sequence-dependent conformational preferences have also been observed in computational studies11 , 21 , 42 and, near recently, analysis of hydroxyl radical cleavage patterns shows that DNA shape is under evolutionary selection43. Such observations suggest that the role of Dna shape must exist taken into consideration when annotating entire genomes and predicting transcription cistron binding sites. The biophysical insights described here, together with the increased availability of high-throughput bounden information, offering the hope of major progress in understanding how proteins recognize specific Deoxyribonucleic acid sequences and in the development of improved predictive algorithms.
Methods Summary
Minor groove geometry was analyzed with Curves44 for all one,031 crystal structures of protein-DNA complexes in the PDB that have any amino acid contacting base of operations atoms. Protein side chains contact the minor groove in 69% of those structures that accept at least one helical plough of DNA. The probabilities for each amino acid to contact the minor groove were calculated for three groups of DNAs: full, narrow, and not narrow. Proteins were grouped based on 40% sequence identity. The backdrop of free DNAs and DNAs spring to proteins were analyzed based on the minor groove widths of tetranucleotides, defined at the central base pair pace.
All 35 crystal structures of the nucleosome available in the PDB were analyzed. The analysis of nucleosomal DNA is based on 23,076 sequences in an in vivo yeast dataset29. The point for a sequence motif in nucleosomal Deoxyribonucleic acid is positive for a base pair when the base pair comprises any part of the sequence motif. Frequencies were symmetrized by analyzing both complementary Deoxyribonucleic acid strands.
Electrostatic potentials were obtained from solutions to the non-linear Poisson-Boltzman equation at physiologic ionic strength using the DelPhi program31 , 45. Regions inside the molecular surface of the Deoxyribonucleic acid were assigned a dielectric constant of 2 while the solvent was assigned a value of 80. The potential is reported at a reference point at the heart of the minor groove. The reference point is located close to the bottom of the groove in approximately the plane of a base pair. This definition provides a measure of electrostatic potential equally a function of base of operations sequence. Solvation costless energies of amino acids were calculated for extended conformations of arginine and lysine side chains and compared for four dissimilar force fields.
Methods
Calculation of minor groove width
In that location were in full ane,031 crystal structures of protein-Deoxyribonucleic acid complexes in the PDB equally of June 1, 2008 in which the DNA was contacted by any amino acid side concatenation at a distance <6.0 Å from base of operations atoms. Of these structures, 567 independent at least ane helical turn, and no chemical modifications or deformations that prevent the calculation of minor groove width. Groove geometry was analyzed using Curves44 and minor groove width was calculated every bit a part of base sequence by averaging all the Curves levels given for each nucleotide.
Statistical assay of protein-Deoxyribonucleic acid contacts
Of the 567 protein-DNA structures in our dataset, 392 have at least one minor groove contact defined by a distance of <six.0 Å between any base and side concatenation atoms. To avoid an oversampling bias, proteins in this dataset that shared ≥40% sequence identity were grouped to create 109 groups. The average number of contacts within each group was subsequently averaged over all 109 groups. These averages were divided by the sum of the average number of contacts for all amino acids to summate the total pocket-size groove contacts, contacts in non narrow modest grooves (≥v.0 Å), and contacts in narrow pocket-sized grooves (<5.0 Å), for each amino acid.
Hydrogen bail contacts between amino acid side chains and the DNA bases and phosphates, water molecules, and other poly peptide atoms were identified with the HBplus program46.
Structural annotation of Deoxyribonucleic acid-bounden proteins
The proteins in our dataset of poly peptide-Dna complexes were classified in SCOP47 superfamilies. Proteins for which SCOP annotations were not available were annotated manually or using the ASTRAL database48.
Correlation of tetranucleotide structure and sequence
Tetranucleotides in free Dna and protein-DNA complexes were used to analyze the base sequence propensity of minor groove regions as a role of minor groove width. The minor groove width of a tetranucleotide was divers by the average of all Curves44 levels for groove width of the second nucleotide and the first level of the third nucleotide, which describes groove width at the central base pair footstep. Cease regions and irregular tetranucleotides were excluded by requiring groove width definitions for at to the lowest degree ane Curves level of each of the four nucleotides. Tetranucleotides from nucleosomal Deoxyribonucleic acid were excluded from this analysis because the Dna is strongly plain-featured and the spacing between narrow regions is fixed at well-nigh 1 helical turn, thus adding a bias to the results. When applied to the 521 protein-DNA complexes in our dataset, these criteria allowed the assay of all 136 possible unique tetranucleotides. When applied to the 88 free DNA structures in our dataset, the same criteria resulted in the assay of 59 unique tetranucleotides. In guild to increase coverage for the free DNA dataset, NMR structures were included if dipolar coupling data were used in the refinement.
Propensity of sequence motifs in nucleosomes
The structural analysis of nucleosomes includes all 35 crystal structures in the PDB as of May 1, 2009. The sequence analysis was based on 23,076 nucleosome sequences of length 146–148 base pairs in a yeast in vivo dataset29. These nucleosome sites were scanned for sequence motifs such equally A-tracts of different length, TpA steps, or other AT- rich regions. A given motif contributed to a positive signal for whatever base pair that overlapped that motif, thus longer motifs contributed signals to more nucleotide positions. The frequencies of all motifs were symmetrized past analyzing both complementary strands.
Calculations of electrostatic potentials
Electrostatic potentials were obtained from solutions to the non-linear Poisson- Boltzman equation at 0.145 M salt using the DelPhi program31 , 45. Partial charges and atomic radii were taken from the Amber force field49. The interior of the molecular surface of the solute molecule (calculated with a 1.iv Å probe sphere) was assigned a dielectric constant of ε=2 while the exterior aqueous phase was assigned a value of ε=80. Debye-Hückel boundary conditions and five focusing steps were used with a cubic grid size of 165 (a grid size of 185 was used for the nucleosome).
The electrostatic potential is reported at a reference betoken close to the bottom of the pocket-size groove approximately in the plane of base of operations pair i. The reference point i is divers as the geometric midpoint between the O4' atoms of nucleotide i+1 in the 5'-3' strand and nucleotide i−1 in the 3'−5' strand12. Where the Deoxyribonucleic acid strongly bends into the major groove the reference point can clash with the guanine amino group and crusade large positive potentials (equally seen in Figure 4a for three regions of the nucleosome).
Desolvation free energies were calculated with the DelPhi programme31 , 45 for the transfer of arginine and lysine side chains in extended conformations from water to a medium of dielectric constant ε=two. Transfer gratuitous energies were calculated for each of the two side chains based on charge distributions and atomic radii taken from Amber49 and 3 other force fields (see Supplementary Table iii).
Supplementary Material
1
Acknowledgments
This work was supported by NIH grants GM54510 (R.Due south.M.) and U54 CA121852 (B.H. and R.S.M.). The authors thank Andrea Califano for many helpful conversations.
Footnotes
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Why Do Rna Read Major Groove and Not Minor Groove on Dna
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2793086/