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At least 397 records · Page 22Linked to original sources
PubMed2000

The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers.

It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity. The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.

Bacteriophage lambda↗
PubMed2000

Backbone dynamics of a cbEGF domain pair in the presence of calcium.

Calcium binding (cb) epidermal growth factor-like (EGF) domains are found in a wide variety of extracellular proteins with diverse functions. In several proteins, including the fibrillins (1 and 2), the low-density lipoprotein receptor, the Notch receptor and related molecules, these domains are organised as multiple tandem repeats. The functional importance of calcium-binding by EGF domains has been underscored by the identification of missense mutations associated with defective calcium-binding, which have been linked to human diseases. Here, we present (15)N backbone relaxation data for a pair of cbEGF domains from fibrillin-1, the defective protein in the Marfan syndrome. The data were best fit using a symmetric top model, confirming the extended conformation of the cbEGF domain pair. Our data demonstrate that calcium plays a key role in stabilising the rigidity of the domain pair on the pico- to millisecond time-scale. Strikingly, the most dynamically stable region of the construct is centred about the domain interface. These results provide important insight into the properties of intact fibrillin-1, the consequences of Marfan syndrome causing mutations, and the ultrastructure of fibrillins and other extracellular matrix proteins.

Amino Acid Sequence↗
PubMed2000

Structure of the utrophin actin-binding domain bound to F-actin reveals binding by an induced fit mechanism.

Utrophin is a large ubiquitously expressed cytoskeletal protein, homologous to dystrophin, the protein disrupted in Duchenne muscular dystrophy. The association of both proteins with the actin cytoskeleton is functionally important and is mediated by a domain at their N termini, conserved in members of the spectrin superfamily, including alpha-actinin, beta-spectrin and fimbrin. We present the structure of the actin-binding domain of utrophin in complex with F-actin, determined by cryo-electron microscopy and helical reconstruction, and a pseudo-atomic model of the complex, generated by docking the crystal structures of the utrophin domain and F-actin into the reconstruction. In contrast to the model of actin binding proposed for fimbrin, the utrophin actin-binding domain appears to associate with actin in an extended conformation. This conformation places residues that are highly conserved in utrophin and other members of the spectrin superfamily at the utrophin interface with actin, confirming the likelihood of this binding orientation. This model emphasises the importance of protein flexibility in modeling interactions and presents the fascinating possibility of a diversity of actin-binding mechanisms among related proteins.

Actins↗
PubMed2000

The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein.

The major packaging signal of human immunodeficiency virus type 1 (HIV-1) RNA has been localised to the region 3' to the major splice donor within the leader sequence. Secondary structural studies for this region of the HIV-1 genome have shown the existence of a stem-loop structure capped by a purine-rich tetraloop. Extensive mapping data presented here lead to the complete characterisation of the structure of the stem-loop, including a new purine-rich internal loop in the lower part of the structure and the previously established GGAG tetraloop at its tip. Biochemical analysis reveals that both internal loop and tetraloop are primary sites for interaction with Gag polyprotein, and that binding of Gag protein leads to a conformational change which alters the RNA structure. NMR spectroscopy has been used to determine the three-dimensional structure of this complete stem-loop structure. The structural analysis reveals a significant difference between the apical part of the stem-loop structure, which adopts a well-defined conformation, and the purine-rich internal loop, which is instead very flexible. In contrast to what is generally observed for internal loop structures in RNA, this region of the encapsidation signal adopts a structure lacking stable interstrand interactions capable of stabilising a unique conformation. We suggest that the stem-loop structure represents a nucleation site for Gag protein binding, and that the protein exploits the flexibility of the internal loop to initiate the unwinding of the structure with successive addition of Gag molecules interacting with the RNA and each other through conserved I (interaction) domains.

Amino Acid Sequence↗
PubMed2000

Experimental and computational studies of the G[UUCG]C RNA tetraloop.

In prokaryotic ribosomal RNAs, most UUCG tetraloops are closed by a C-G base-pair. However, this preference is greatly reduced in eukaryotic rRNA species where many UUCG tetraloops are closed by G-C base-pairs. Here, biophysical properties of the C[UUCG]G and G[UUCG]C tetraloops are compared, using experimental and computational methods. Thermal denaturation experiments are used to derive thermodynamic parameters for the wild-type G[UUCG]C tetraloop and variants containing single deoxy substitutions in the loop. A comparison with analogous experiments on the C[UUCG]G motif shows that the two RNA species exhibit similar patterns in response to the substitutions, suggesting that their loop structures are similar. This conclusion is supported by NMR data that suggest that the essential UUCG loop structure is maintained in both tetraloops. However, NMR results show that the G[UUCG]C loop structure is disrupted prior to melting of the stem; this behavior is in contrast to the two-state behavior of the C[UUCG]G molecule. Stochastic dynamics simulations using the GB/SA continuum solvation model, run as a function of temperature, show rare conformational transitions in several G[UUCG]C simulations. These results lead to the conclusion that substitution of a G-C for a C-G closing base-pair increases the intrinsic flexibility of the UUCG loop.

Bacteria↗
PubMed2000

The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex.

Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented. The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker. The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N-terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide. The structure of a mutant beta polypeptide W(+9)F was also determined. This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding. Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane. We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1. This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes.

Amino Acid Sequence↗
PubMed2000

Mapping the protein-DNA interface and the metal-binding site of the major human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic (AP) endonuclease Ape1 is a key enzyme in the mammalian base excision repair pathway that corrects AP sites in the genome. Ape1 cleaves the phosphodiester bond immediately 5' to AP sites through a hydrolytic reaction involving a divalent metal co-factor. Here, site-directed mutagenesis, chemical footprinting techniques, and molecular dynamics simulations were employed to gain insights into how Ape1 interacts with its metal cation and AP DNA. It was found that Ape1 binds predominantly to the minor groove of AP DNA, and that residues R156 and Y128 contribute to protein-DNA complex stability. Furthermore, the Ape1-AP DNA footprint does not change along its reaction pathway upon active-site coordination of Mg(2+) or in the presence of DNA polymerase beta (polbeta), an interactive protein partner in AP site repair. The DNA region immediately 5' to the abasic residue was determined to be in close proximity to the Ape1 metal-binding site. Experimental evidence is provided that amino acid residues E96, D70, and D308 of Ape1 are involved in metal coordination. Molecular dynamics simulations, starting from the active site of the Ape1 crystal structure, suggest that D70 and E96 bind directly to the metal, while D308 coordinates the cation through the first hydration shell. These studies define the Ape1-AP DNA interface, determine the effect of polbeta on the Ape1-DNA interaction, and reveal new insights into the Ape1 active site and overall protein dynamics.

Amino Acid Substitution↗
PubMed2000

Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies.

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.

Animals↗
PubMed2000

High-resolution structure of the OmpA membrane domain.

The membrane domain of OmpA consists of an eight-stranded all-next-neighbor antiparallel beta-barrel with short turns at the periplasmic barrel end and long flexible loops at the external end. The structure analysis has been extended from medium resolution to 1. 65 A (1 A=0.1 nm), and the molecular model has been refined anisotropically to show oriented mobilities of the structural elements. The improved data allowed us to locate five further detergent molecules and 11 more water molecules. Moreover, the two large non-polar packing contacts have now been defined in detail. The analysis indicates that the beta-barrel constitutes a solid scaffold such that the long external loops need not contribute to stability. These loops are highly mobile and thus cause a major problem during the crystallization process. The beta-barrel was related to those of lipocalins. Two further crystal forms with exceptionally dense packing arrangements were established at medium resolution.

Amino Acid Substitution↗
PubMed2000

Microtubules switch occasionally into unfavorable configurations during elongation.

Tubulin assembles to form a range of structures that differ by their protofilament and monomer helix-start numbers. The microtubule lattice is believed to accommodate these different configurations by skewing the protofilaments so that the lateral interactions between tubulin subunits are maintained. Here, we present the characterization of 14 types of microtubules, including six novel ones, through an extensive analysis of microtubules assembled in vitro from pure tubulin. Although the six new types represented only 1 % of the total length of the population examined ( approximately 17 mm), they define the limits of microtubule structure and assembly. Protofilament skewing is restricted to within +/-2 degrees. Outside this range, the restoring force induced by the skewed protofilaments is compensated by a longitudinal shift (less than +/-0.2 nm) between adjacent protofilaments. Configurations with theoretical protofilament skew angles larger than +/-4 degrees or that necessitate larger modifications of the microtubule surface lattice were not observed. Analysis of the microtubule types distribution reveals that it is sharply peaked around the less skewed conformations. These results indicate that both the flexibility of the protofilaments and the strength of their lateral interactions restrict the range of structures assembled. They also demonstrate that growing microtubules can occasionally switch into energetically unfavorable configurations, a behavior that may account for the stochastic nature of catastrophes.

Animals↗
PubMed2000

Stabilization of GroEL minichaperones by core and surface mutations.

We report the crystal structures of two hexa-substituted mutants of a GroEL minichaperone that are more stable than wild-type by 7.0 and 6.1 kcal mol(-1). Their structures imply that the increased stability results from multiple factors including improved hydrophobic packing, optimised hydrogen bonding and favourable structural rearrangements. It is commonly believed that protein core residues are immutable and generally optimized for energy, while on the contrary, surface residues are variable and hence unimportant for stability. But, it is now becoming clear that mutations of both core and surface residues can increase protein stability, and that protein cores are more flexible and thus more tolerant to mutation than expected. Sequence comparison of homologous proteins has provided a way to pinpoint the residues that contribute constructively to stability and to guide the engineering of protein stability. Stabilizing mutations identified by this approach are most frequently located at protein surfaces but with a few found in protein cores. In the latter case, local flexibility in the hydrophobic core is the key factor that allows the energetically favourable burial of larger hydrophobic side-chains without undue energetic penalties from steric clashes.

Amino Acid Substitution↗
PubMed2000

Cross-linking constraints on F-actin structure.

The DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274), and the C terminus region are among the structural elements of monomeric (G-) actin proposed to form the intermonomer interface in F-actin. To test the proximity and interactions of these elements and to provide constraints on models of F-actin structure, cysteine residues were introduced into yeast actin either at residue 41 or 265. These mutations allowed for specific cross-linking of F-actin between C41 and C265, C265 and C374, and C41 and C265 using dibromobimane and disulfide bond formation. The cross-linked products were visualized on SDS-PAGE and by electron microscopy. Model calculations carried out for the cross-linked F-actins revealed that considerable flexibility or displacement of actin residues is required in the disulfide cross-linked segments to fit these filaments into model F-actin structures. The calculated, cross-linked structures showed a better fit to the Holmes rather than the refined Lorenz model of F-actin. It is predicted on the basis of such calculations that image reconstruction of electron micrographs of disulfide cross-linked C41-C374 F-actin should provide a conclusive test of these two similar models of F-actin structure.

Actins↗
PubMed2000

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs.

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.

Animals↗
PubMed2000

Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module.

Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.

Amino Acid Sequence↗
PubMed2000

Backbone dynamics of the C-terminal SH2 domain of the p85alpha subunit of phosphoinositide 3-kinase: effect of phosphotyrosine-peptide binding and characterization of slow conformational exchange processes.

The backbone dynamics of the C-terminal SH2 domain from the regulatory subunit p85alpha (p85alpha C-SH2) of phosphoinositide 3-kinase has been investigated in the absence of, and in complex with, a high-affinity phosphotyrosine-containing peptide ligand derived from the platelet-derived growth-factor receptor. (15)N R(1) and R(2) relaxation rates and steady-state [(1)H]-(15)N NOE values were measured by means of (1)H-(15)N correlated two-dimensional methods and were analyzed within the framework of the model-free formalism. Several residues in the BC loop and in the neighbouring secondary structural elements display fast local dynamics in the absence of phosphotyrosine peptide ligand as evidenced by below-average [(1)H]-(15)N NOE values. Furthermore, residue Gln41 (BC3) displays conformational exchange phenomena as indicated by an above-average R(2) relaxation rate. Upon binding of the phosphotyrosine peptide, the NOE values increase to values observed for regular secondary structure and the exchange contribution to the R(2) relaxation rate for Gln41 (BC3) vanishes. These observations indicate a loss of backbone flexibility upon ligand binding. Substantial exchange contributions for His56 (betaD4) and Cys57 (betaD5), which are known to make important interactions with the ligand, are attenuated upon ligand binding. Several residues in the betaD'-FB region and the BG loop, which contribute to the ligand binding surface of the protein, exhibit exchange terms which are reduced or vanish when the ligand is bound. Together, these observations suggest that ligand binding is accompanied by a loss of conformational flexibility on the ligand binding face of the protein. However, comparison with other SH2 domains reveals an apparent lack of consensus in the changes in dynamics induced by ligand binding. Exchange rates for individual residues were quantified in peptide-complexed p85alpha C-SH2 from the dependence of the exchange contributions on the CPMG delay in an R(2) series and show that peptide-complexed p85alpha C-SH2 is affected by multiple conformational exchange processes with exchange rate constants from 10(2) s(-1) to 7.10(3) s(-1). Mapping of the exchange-rate constants on the protein surface show a clustering of residues with similar exchange-rate constants and suggests that clustered residues are affected by a common predominant exchange process.

Amino Acid Sequence↗
PubMed2000

A DNA structural atlas for Escherichia coli.

We have performed a computational analysis of DNA structural features in 18 fully sequenced prokaryotic genomes using models for DNA curvature, DNA flexibility, and DNA stability. The structural values that are computed for the Escherichia coli chromosome are significantly different from (and generally more extreme than) that expected from the nucleotide composition. To aid this analysis, we have constructed tools that plot structural measures for all positions in a long DNA sequence (e.g. an entire chromosome) in the form of color-coded wheels (http://www.cbs.dtu. dk/services/GenomeAtlas/). We find that these "structural atlases" are useful for the discovery of interesting features that may then be investigated in more depth using statistical methods. From investigation of the E. coli structural atlas, we discovered a genome-wide trend, where an extended region encompassing the terminus displays a high of level curvature, a low level of flexibility, and a low degree of helix stability. The same situation is found in the distantly related Gram-positive bacterium Bacillus subtilis, suggesting that the phenomenon is biologically relevant. Based on a search for long DNA segments where all the independent structural measures agree, we have found a set of 20 regions with identical and very extreme structural properties. Due to their strong inherent curvature, we suggest that these may function as topological domain boundaries by efficiently organizing plectonemically supercoiled DNA. Interestingly, we find that in practically all the investigated eubacterial and archaeal genomes, there is a trend for promoter DNA being more curved, less flexible, and less stable than DNA in coding regions and in intergenic DNA without promoters. This trend is present regardless of the absolute levels of the structural parameters, and we suggest that this may be related to the requirement for helix unwinding during initiation of transcription, or perhaps to the previously observed location of promoters at the apex of plectonemically supercoiled DNA. We have also analyzed the structural similarities between groups of genes by clustering all RNA and protein-encoding genes in E. coli, based on the average structural parameters. We find that most ribosomal genes (protein-encoding as well as rRNA genes) cluster together, and we suggest that DNA structure may play a role in the transcription of these highly expressed genes.

Bacterial Proteins↗
PubMed2000

Aspartyl tRNA-synthetase from Escherichia coli: flexibility and adaptability to the substrates.

The crystal structure of aspartyl-tRNA synthetase from Escherichia coli has been determined to a resolution of 2.7 A. The structure is compared to the same enzyme co-crystallized with tRNA(Asp) and containing aspartyl adenylate or ATP. The asymmetric unit contains three monomers of the enzyme. While most parts of the protein show no significant differences in the three monomers, a few regions cannot be superimposed. Those regions are characterized by a high B-factor, and consist mostly of loops that make contacts with the tRNA in the complexes. The flexibility of the protein is seen at a global level, by the observation of a 10 to 15 degrees rotation of the N-terminal and insertion domains upon tRNA binding, and at the level of the individual amino acid residues, by main-chain and side-chain rearrangements. In contrast to these induced-fit conformational changes, a few residues essential for the tRNA anticodon or aspartyl-adenylate recognition exist in a predefined conformation, ensured by specific interactions within the protein.

Adenosine Monophosphate↗
PubMed2000

Signals for TBP/TATA box recognition.

The TATA box-binding protein (TBP) recognizes its target sites (TATA boxes) by indirectly reading the DNA sequence through its conformation effects (indirect readout). Here, we explore the molecular mechanisms underlying indirect readout of TATA boxes by TBP by studying the binding of TBP to adenovirus major late promoter (AdMLP) sequence variants, including alterations inside as well as in the sequences flanking the TATA box. We measure here the dissociation kinetics of complexes of TBP with AdMLP targets and, by phase-sensitive assay, the intrinsic bending in the TATA box sequences as well as the bending of the same sequence induced by TBP binding. In these experiments we observe a correlation of the kinetic stability to sequence changes within the TATA recognition elements. Comparison of the kinetic data with structural properties of TATA boxes in known crystalline TBP/TATA box complexes reveals several "signals" for TATA box recognition, which are both on the single base-pair level, as well as larger DNA tracts within the TATA recognition element. The DNA bending induced by TBP on its binding sites is not correlated to the stability of TBP/TATA box complexes. Moreover, we observe a significant influence on the kinetic stability of alteration in the region flanking the TATA box. This effect is limited however to target sites with alternating TA sequences, whereas the AdMLP target, containing an A tract, is not influenced by these changes.

Adenoviridae↗