Indirect Readout Mechanism Research Articles

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor: THE ROLE OF DNA DEFORMATION ENERGY

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.

Alteration of Sequence Specificity of the Type II Restriction Endonuclease HincII through an Indirect Readout Mechanism

The functional and structural consequences of a mutation of the DNA intercalating residue of HincII, Q138F, are presented. Modeling has suggested that the DNA intercalation by Gln-138 results in DNA distortions potentially used by HincII in indirect readout of its cognate DNA, GTYRAC (Y = C or T, R = A or G) (Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47). Kinetic data presented here indicate that the mutation of glutamine 138 to phenylalanine (Q138F) results in a change in sequence specificity at the center two base pairs of the cognate recognition site. We show that the preference of HincII for cutting, but not binding, the three cognate sites differing in the center two base pairs has been altered by the mutation Q138F. Five new crystal structures are presented including Q138F HincII bound to GTTAAC and GTCGAC both with and without Ca2+ as well as the structure of wild type HincII bound to GTTAAC. The Q138F HincII/DNA structures show conformational changes in the protein, bound DNA, and at the protein-DNA interface, consistent with the formation of adaptive complexes. Analysis of these structures and the effect of Ca2+ binding on the protein-DNA interface illuminates the origin of the altered specificity by the mutation Q138F in the HincII enzyme.

Structure of the S-adenosylmethionine riboswitch regulatory mRNA element

Riboswitches are cis-acting genetic regulatory elements found in the 5'-untranslated regions of messenger RNAs that control gene expression through their ability to bind small molecule metabolites directly. Regulation occurs through the interplay of two domains of the RNA: an aptamer domain that responds to intracellular metabolite concentrations and an expression platform that uses two mutually exclusive secondary structures to direct a decision-making process. In Gram-positive bacteria such as Bacillus species, riboswitches control the expression of more than 2% of all genes through their ability to respond to a diverse set of metabolites including amino acids, nucleobases and protein cofactors. Here we report the 2.9-angstroms resolution crystal structure of an S-adenosylmethionine (SAM)-responsive riboswitch from Thermoanaerobacter tengcongensis complexed with S-adenosylmethionine, an RNA element that controls the expression of several genes involved in sulphur and methionine metabolism. This RNA folds into a complex three-dimensional architecture that recognizes almost every functional group of the ligand through a combination of direct and indirect readout mechanisms. Ligand binding induces the formation of a series of tertiary interactions with one of the helices, serving as a communication link between the aptamer and expression platform domains.

Sequence-Dependent Conformational Energy of DNA Derived from Molecular Dynamics Simulations: Toward Understanding the Indirect Readout Mechanism in Protein−DNA Recognition

Sequence dependence of DNA conformation plays a crucial role in its recognition by proteins and ligands. To clarify the relationship between sequence and conformation, it is necessary to quantify the conformational energy and specificity of DNA. Here, we make a systematic analysis of dodecamer DNA structures including all the 136 unique tetranucleotide sequences at the center by molecular dynamics simulations. Using a simplified conformational model with six parameters to describe the geometry of adjacent base pairs and harmonic potentials along these coordinates, we estimated the equilibrium conformational parameters and the harmonic potentials of mean force for the central base-pair steps from many trajectories of the simulations. This enabled us to estimate the conformational energy and the specificity for any given DNA sequence and structure. We tested our method by using sequence-structure threading to estimate the conformational energy and the Z-score as a measure of specificity for many B-DNA and A-DNA crystal structures. The average Z-scores were negative for both kinds of structures, indicating that the potential of mean force from the simulation is capable of predicting sequence specificity for the crystal structures and that it may be used to study the sequence specificity of both types of DNA. We also estimated the positional distribution of conformational energy and Z-score within DNA and showed that they are strongly position dependent. This analysis enabled us to identify particular conformations responsible for the specificity. The presented results will provide an insight into the mechanisms of DNA sequence recognition by proteins and ligands.

The Role of DNA Structure and Dynamics in the Recognition of Bovine Papillomavirus E2 Protein Target Sequences

The papillomavirus E2 transcription and replication factors bind to the DNA consensus ACCGN4CGGT sequence (E2-BS), through both direct and indirect readout mechanisms. The two symmetric half-sites ACCG·CGGT are highly conserved in the genomes and are hydrogen bound with E2. Although E2 does not contact the N4 spacer, the affinities are modulated by the base composition of this DNA part. Nevertheless, the origin of either the global recognition mechanism or the spacer effect remains unclear, particularly in the case of the bovine papillomavirus type 1 E2 (BPV-1-E2) system, used as model to study the papillomaviruses. We present, herein, studies carried out on oligomers differently recognized by the BPV-1-E2 protein and based on molecular dynamic simulations including counterions and water. The sequences contain the conserved half-sites but three different spacers (CCAT, ACGT and AAAC), resulting in very high, high and low affinity targets for BPV-1-E2. In order to estimate how much the free DNAs resemble the bound conformations, comparisons are made with two DNAs extracted from E2-BS-BPV-1 crystallographic complexes, representative of high and moderate affinity structures. The analysis of 15 ns trajectories reveals that the ACCG/CGGT half-sites, whatever the spacer, have the same behavior and adopt average stable base-pair parameters very close to those of the bound conformations. In contrast, the three different free spacers strongly differ in their BI↔BII backbone dynamics. The low affinity AAAC spacer exhibits stable BI backbone conformations, the high affinity ACGT spacer is characterized by a dramatic instability of the CpG phosphate groups, and the CpA and GpG backbones in the very high affinity CCAT·ATGG spacer are trapped in BII conformations. All resemble more of the moderate affinity complex DNA than the high affinity one. Nevertheless, the particular behavior of the CCAT and ACGT backbones allows the emergence of BII-rich spacers, a configuration reproducing both local and global helical features of the bound DNA conformation of the high affinity complex and favoring the minor groove curvature required in the complex. In particular, the CCAT-containing site spends almost half of the time in this form that well mimics the bound one. Thus, we propose that the E2 protein could take advantage of the invariant favorable structures of the half-sites to form a pre-complex, but would require a specific spacer intrinsic malleability to lock the interaction. Finally, the backbone conformational states, by their ability to translate information coded in the sequence into structural properties, provide insight into the mechanisms that contribute to fine binding site selection and specific nucleic acid ligand recognition.

The Role of DNA Structure and Dynamics in the Recognition of Bovine Papillomavirus E2 Protein Target Sequences

The papillomavirus E2 transcription and replication factors bind to the DNA consensus ACCGN4CGGT sequence (E2-BS), through both direct and indirect readout mechanisms. The two symmetric half-sites ACCG·CGGT are highly conserved in the genomes and are hydrogen bound with E2. Although E2 does not contact the N4 spacer, the affinities are modulated by the base composition of this DNA part. Nevertheless, the origin of either the global recognition mechanism or the spacer effect remains unclear, particularly in the case of the bovine papillomavirus type 1 E2 (BPV-1-E2) system, used as model to study the papillomaviruses. We present, herein, studies carried out on oligomers differently recognized by the BPV-1-E2 protein and based on molecular dynamic simulations including counterions and water. The sequences contain the conserved half-sites but three different spacers (CCAT, ACGT and AAAC), resulting in very high, high and low affinity targets for BPV-1-E2. In order to estimate how much the free DNAs resemble the bound conformations, comparisons are made with two DNAs extracted from E2-BS-BPV-1 crystallographic complexes, representative of high and moderate affinity structures. The analysis of 15ns trajectories reveals that the ACCG/CGGT half-sites, whatever the spacer, have the same behavior and adopt average stable base-pair parameters very close to those of the bound conformations. In contrast, the three different free spacers strongly differ in their BI↔BII backbone dynamics. The low affinity AAAC spacer exhibits stable BI backbone conformations, the high affinity ACGT spacer is characterized by a dramatic instability of the CpG phosphate groups, and the CpA and GpG backbones in the very high affinity CCAT·ATGG spacer are trapped in BII conformations. All resemble more of the moderate affinity complex DNA than the high affinity one. Nevertheless, the particular behavior of the CCAT and ACGT backbones allows the emergence of BII-rich spacers, a configuration reproducing both local and global helical features of the bound DNA conformation of the high affinity complex and favoring the minor groove curvature required in the complex. In particular, the CCAT-containing site spends almost half of the time in this form that well mimics the bound one. Thus, we propose that the E2 protein could take advantage of the invariant favorable structures of the half-sites to form a pre-complex, but would require a specific spacer intrinsic malleability to lock the interaction. Finally, the backbone conformational states, by their ability to translate information coded in the sequence into structural properties, provide insight into the mechanisms that contribute to fine binding site selection and specific nucleic acid ligand recognition.

A model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility

Initial recognition of DNA damage is the crucial but poorly understood first step in DNA repair by the human nucleotide excision repair (NER) and mismatch repair (MMR) systems. Failure by NER or MMR to recognize DNA damage threatens the genetic integrity of the organism and may play a role in carcinogenesis. Both NER and MMR recognize and repair a wide variety of structurally dissimilar lesions against the background of normal DNA. Previous studies have suggested that detection of thermodynamic destabilization of DNA caused by covalent damage and base mismatches is a potential mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates. However, both NER and MMR respectively, repair a wide variety of stabilizing and destabilizing covalent DNA lesions and base pair mismatches. A common feature of lesions that are both thermodynamically stabilizing and destabilizing is the alteration of the local DNA flexibility (dynamics). In this review we describe the experimental evidence for altered dynamics from NMR and thermodynamic studies on normal and damaged DNA molecules with respect to recognition by NER and MMR. Based on these data, we propose a model for initial detection of lesions by both NER and MMR that occurs through an indirect readout mechanism of alternative DNA conformations induced by covalent damage and base mismatches.

Intermolecular and Intramolecular Readout Mechanisms in Protein–DNA Recognition

Protein–DNA recognition plays an essential role in the regulation of gene expression. Regulatory proteins are known to recognize specific DNA sequences directly through atomic contacts (intermolecular readout) and/or indirectly through the conformational properties of the DNA (intramolecular readout). However, little is known about the respective contributions made by these so-called direct and indirect readout mechanisms. We addressed this question by making use of information extracted from a structural database containing many protein–DNA complexes. We quantified the specificity of intermolecular (direct) readout by statistical analysis of base–amino acid interactions within protein–DNA complexes. The specificity of the intramolecular (indirect) readout due to DNA was quantified by statistical analysis of the sequence-dependent DNA conformation. Systematic comparison of these specificities in a large number of protein–DNA complexes revealed that both intermolecular and intramolecular readouts contribute to the specificity of protein–DNA recognition, and that their relative contributions vary depending upon the protein–DNA complexes. We demonstrated that combination of the intermolecular and intramolecular energies derived from the statistical analyses lead to enhanced specificity, and that the combined energy could explain experimental data on binding affinity changes caused by base mutations. These results provided new insight into the relationship between specificity and structure in the process of protein–DNA recognition, which would lead to prediction of specific protein–DNA binding sites.

Base Coupling in Sequence-specific Site Recognition by the ETS Domain of Murine PU.1

The ETS domain of murine PU.1 tolerates a large number of DNA cognates bearing a central consensus 5′-GGAA-3′ that is flanked by a diverse combination of bases on both sides. Previous attempts to define the sequence selectivity of this DNA binding domain by combinatorial methods have not successfully predicted observed patterns among in vivo promoter sequences in the genome, and have led to the hypothesis that energetic coupling occurs among the bases in the flanking sequences. To test this hypothesis, we determined, using thermodynamic cycles, the complex stabilities and base coupling energies of the PU.1 ETS domain for a set of 26 cognate variants (based on the λB site of the Igλ2-4 enhancer, 5′- AATAAAA GGAA GTGAAACCAA-3′ ) in which flanking sequences up to three bases upstream and/or two bases downstream of the core consensus are substituted. We observed that both cooperative and anticooperative coupling occurs commonly among the flanking sequences at all the positions investigated. This phenomenon extends at least three bases in the 5′ side and is, at least on our experimental data, due exclusively to pairwise interactions between the flanking bases, and not changes in the local environment of the DNA groove floor. Energetic coupling also occurs between the flanking sides across the core consensus, suggesting long-range conformational effects along the DNA target and/or in the protein. Our data provide an energetic explanation for the pattern of flanking bases observed among in vivo promoter sequences and reconcile the apparent discrepancies raised by the combinatorial experiments. We also discuss the significance of base coupling in light of an indirect readout mechanism in ETS/DNA site recognition.

The tRNA specificity of Thermus thermophilus EF-Tu.

By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)*GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from -11.7 kcal/mol for Val-tRNA(Glu) to -8.1 kcal/mol for Val-tRNA(Tyr), clearly establishing EF-Tu*GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of k(off) varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism.

Target Prediction of Transcription Factors: Refinement of Structure-Based Method

Gene regulation in higher organisms is achieved by a complex system of transcription factors. Complete genome sequences of many organisms have opened up the possibility of systematic analysis of gene regulation at the genome level. Transcription factors usually bind to multiple target sequences and regulate multiple genes in a complex manner. Because there are a large number of transcription factors and their targets, it will be difficult to analyze such system by experiment alone. Bioinformatics should play an important role in elucidating the mechanism of gene regulation. In particular, finding target genes for transcription factors at the genome level will lay a basis for the analysis of gene regulatory network. We have been developing methods for predicting target sequences of transcription factors. Structure-based method, which utilizes structural data of protein-DNA complexes, is one of the promising methods for the target prediction as reported earlier. Here we describe the refinement of the structure-based methods by inclusion of in-direct readout as well as direct readout mechanisms in protein-DNA recognition. We show that both the direct and in-direct readout mechanisms contribute to the specificity of protein-DNA recognition significantly, and that the combination of the two mechanisms will increase the accuracy of target prediction.

DNA Binding Specificity Studies of Four ETS Proteins Support an Indirect Read-out Mechanism of Protein-DNA Recognition

Members of the ETS family of transcription factors are involved in several developmental and physiological processes, and, when overexpressed or misexpressed, can contribute to a variety of cancers. Each family member has a conserved DNA-binding domain that recognizes DNA sequences containing a G-G-A trinucleotide. Discrimination between potential ETS-binding sites appears to be governed by both the nucleotides flanking the G-G-A sequence and protein-protein interactions. We have used an adaptation of the "length-encoded multiplex" approach (Desjarlais, J. R., and Berg, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11099-11103) to define DNA binding specificities for four ETS proteins: Fli-1, SAP-1, PU.1, and TEL. Our results support a model in which cooperative effects among neighboring bases flanking the central G-G-A site contribute to the formation of stable ETS/DNA complexes. These results are consistent with a mechanism for specific DNA binding that is partially governed by an indirect read-out of the DNA sequence, in which a sequence-specific DNA conformation is sensed or induced.

Investigation of the pyrimidine preference by the c-Myb DNA-binding domain at the initial base of the consensus sequence.

The principal determinant of the pyrimidine preference by the c-Myb DNA-binding domain at the initial base of the consensus sequence was investigated by mutation of both the protein and the DNA base pairs, with analysis by a filter binding assay. Amino acid residue 187 was revealed to interact with the pyrimidine base position, as estimated from our previous complex structure. Unexpectedly, since the pyrimidine preference is retained even in the Gly187 mutant, the principal origin of the base specificity should not occur via the direct-readout mechanism, but by an indirect-readout mechanism, namely in the intrinsic "bendability" of the pyrimidine-purine step of the DNA duplex. A significant but rather small positive base pair roll is detectable in the conformation of DNA in complex with the c-Myb DNA-binding domain. Following the conventional chemical rules of the direct-readout mechanism, amino acid mutagenesis at position 187 yielded several new base preferences for the protein.

Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs.

Single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized DNA-binding domains (DBD) which are connected with a peptide linker in a head-to-tail arrangement. The prototype RR69 contains two wild-type DBDs, while RR*69 contains a wild-type and an engineered DBD. In this latter domain, the DNA- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related P22 repressor. We have used binding site selection, targeted mutagenesis and binding affinity studies to define the optimum DNA recognition sequence for these single-chain proteins. It is shown that RR69 recognizes DNA sequences containing the consensus boxes of the 434 operators in a palindromic arrangement, and that RR*69 optimally binds to non-palindromic sequences containing a 434 operator box and a TTAA box of which the latter is present in most P22 operators. The spacing of these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both single-chain repressors, similar to that of the 434 repressor, is influenced indirectly by the sequence of the non-contacted, spacer region. Thus, high affinity binding is dependent on both direct and indirect recognition. Nonetheless, the single-chain framework can accommodate certain substitutions to obtain altered DNA-binding specificity and RR*69 represents an example for the combination of altered direct and unchanged indirect readout mechanisms.

Electrostatic analysis of DNA binding properties in lysine to leucine mutants of TATA-box binding proteins.

The structures of the complexes between TATA-box binding proteins (TBPs) and DNA solved recently with X-ray crystallography identify both direct and indirect readout interactions. Examples of indirect readout mechanisms in these complexes are DNA bending and non-local electrostatic complementarity. An intriguing question arising from these structures is the role that a series of lysine residues may have in DNA binding. Thus, in the yeast complex, seven lysines are found to be close to the phosphate backbone, but they appear to form hydrogen bonds to the protein and not to be involved in any direct (or water-mediated) interactions with the DNA. The proposal based on the crystal structure, that these residues set up a delocalized electrostatic potential that stabilizes the complex with DNA, is evaluated here from calculations of the electrostatic potentials generated by the wild-type TBP and various lysine to leucine mutants. The results suggest a grouping of these mutants into three classes, based on their phenotypes and electrostatic profiles. As these groups are affected differently by specific measures taken to rescue DNA binding and transcription functions, the mechanistic inferences from the analysis can be probed experimentally in a manner that also reveals possible binding sites for transcription factors IIA and IIB to the TBP-DNA complex in the transcription preinitiation complex.