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Biomedical subjects

T E Haran

Publications and source records attributed to T E Haran.

14 recordsLinked to original sources
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↗
PubMed1998

Direct versus indirect readout in the interaction of the trp repressor with non-canonical binding sites.

Both direct and indirect readouts are utilized when the trp repressor binds to its operators. Here, we use gel-electrophoretic methods to examine the role played by direct and indirect readouts in the interaction of the repressor with a non-canonical binding site, similar to the mtr operator, and named trpGG. The stability and affinity of the 1:1 complexes of the trp repressor with this non-canonical site are lower than those of the 1:1 complexes formed with either the natural consensus sequence or a consensus sequence found in a selection experiment. We attribute this to the inability of the trpGG target to make the same number of water-mediated hydrogen bonds as canonical trp binding sites. On the other hand, the 2:1 complex of the repressor with trpGG has high stability and affinity, similar to that of the 2:1 complex with a consensus sequence found by a selection experiment. The bend angle induced on the trpGG target by the binding of one repressor molecule is 27 degrees, which is similar to that measured in other 1:1 complexes with the repressor. The angle for the 2:1 complex is significantly larger (43 degrees versus 30 degrees in other 2:1 complexes). We present evidence suggesting that the deleterious effect of the sequence substitution in trpGG is compensated by the increased bend angle in the 2:1 complex. These observations demonstrate that indirect readout may complement for direct readout in determining the nature of the interaction between trp repressor and its binding sites.

Bacterial Proteins↗
PubMed1998

Statistical and structural analysis of trp binding sites: comparison of natural and in vitro selected sequences.

Two different modes can be used when the trp repressor binds to trp binding sites. In the "full-site mode" each repressor molecule is bound to a DNA target containing at least two conserved five base pair tracts separated by eight base pairs. The binding of the repressor to natural trp operators is of this kind. In the "half-site mode" two repressor molecules are sequence-specifically bound, with infinite cooperativity, to two abutting DNA pentamers. We present evidence suggesting that the sequences obtained by a recent in vitro selection assay (Czernik et al. J. Biol. Chem. 269, 27869-27875, 1994) were selected by the binding of two repressor molecules, and that the repressor is bound to most of these sequences using the half-site mode. Using the results of the selection assay, and the set of natural trp binding sites, we characterize the different sequence requirements of the "full-site" versus the "half-site" binding modes. A statistical analysis of the information content of these binding sites shows that functional information on protein binding modes can be extracted from a set of DNA binding sites by comparing the information content of two different DNA populations, or sub-populations. Furthermore, it shows that the binding of proteins to sequences selected by a functional in vitro assay do not necessarily mimic the binding of the protein to the natural targets, even if the information content is similar in the two DNA target populations, i.e., even if the stringency of the selection assay is adequate for locating natural-like sequences. In addition, we show that the structural requirements for protein-DNA interactions can be achieved by different conformations at the base-pair level. Differences in the structural characteristics of different base-pair steps can be used to determine the binding mode and differential binding affinity, which can be utilized in the regulation of several binding sites by a single specific protein.

Apoproteins↗
PubMed1997

X-ray and solution studies of DNA oligomers and implications for the structural basis of A-tract-dependent curvature.

DNA containing short periodic stretches of adenine residues (known as A-tracts), which are aligned with the helical repeat, exhibit a pronounced macroscopic curvature. This property is thought to arise from the cumulative effects of a distinctive structure of the A-tract. It has also been observed by gel electrophoresis that macroscopic curvature is largely retained when inosine bases are introduced singly into A-tracts but decreases abruptly for pure I-tracts. The structural basis of this effect is unknown. Here we describe X-ray and gel electrophoretic analyses of several oligomers incorporating adenine or inosine bases or both. We find that macroscopic curvature is correlated with a distinctive base-stacking geometry characterized by propeller twisting of the base-pairs. Regions of alternating adenine and inosine bases display large propeller twisting comparable to that of pure A-tracts, whereas the values observed for pure I-tracts are significantly smaller. We also observe in the crystal structures that propeller twist leads to close cross-strand contacts between amino groups from adenine and cytosine bases, indicating an attractive NH-N interaction, which is analogous to the NH-O interaction proposed for A-tracts. This interaction also occurs between adenine bases across an A-T step and may explain in part the different behavior of A-T versus T-A steps in the context of A-tract-induced curvature. We also note that hydration patterns may contribute to propeller-twisted conformation. Based on the present data and other structural and biophysical studies, we propose that DNA macroscopic curvature is related to the structural invariance of A-tract and A-tract-like regions conferred by high propeller twist, cross-strand interactions and characteristic hydration. The implications of these findings to the mechanism of DNA bending are discussed.

Adenine↗
PubMed1997

Repressor assembly at trp binding sites is dependent on the identity of the intervening dinucleotide between the binding half sites.

The interaction of trp repressor with its DNA targets is unusual in that specific recognition in this system does not rely exclusively on direct hydrogen bonds to the DNA bases that are crucial for sequence-specific recognition. It has been suggested that trp operators are mainly recognized by water-mediated interactions and by structural recognition of DNA deformability. Here we study the effect of the central dinucleotide on the mode of interaction of the trp repressor with its binding sites. The study was carried out on two consensus sequences: (1) trpTA, the consensus of naturally occurring trp binding sites, containing a T-A step between the two hexameric half-site sequences, ACTAGT; (2) trpAC, a consensus sequence derived from a functional selection study, containing a central A-C step. We show that the identity of the central dinucleotide does not affect the interaction of the first trp repressor molecule with the primary DNA target site, however, it influences the assembly of additional repressor molecules at adjacent sites. Central A-C steps stabilize tandem binding, whereas T-A steps destabilize it. It has been previously suggested that in vivo regulation of trp operators is due to their differential ability to bind multiple repressor molecules. The observations presented here support this model. We ascribe this ability to two sequence-dependent factors which act together: the identity and number of half-site sequences, recognized by water-mediated hydrogen bonds, and the ability of the intervening dinucleotides to form direct bidentate hydrogen bonds to the repressor. Furthermore, we measured the intrinsic and the induced bending of trp operators by the repressor. We find that the operators are straight in their free form, bent by 23 degrees when bound by a single trp repressor molecule, and bent by 30 degrees when bound by two repressor molecules.

Bacterial Proteins↗
PubMed1994

Sequence elements responsible for DNA curvature.

Intrinsic DNA bending or curvature is a phenomenon that has been shown to play an important role in a variety of DNA transactions. Large curvature occurs when short homopolymeric (dA.dT)4-6 runs (A-tracts) are repeated in phase with the helical screw. We have used electrophoretic mobility modulation to examine how bending depends on the nature of the 5 bp DNA sequence between the A tracts in molecules of the form (A5-6N5)n. We show that A-tract-induced DNA curvature can indeed be affected by other sequence elements, although by only about +/- 10%. The small observed curvature modulation implies that the overall helix axis deflection contributed by 5-bp B-DNA segments between A-tracts varies little from one sequence to another. This result validates, to first order, the assumption that DNA curvature results from inserting A-tracts at integral turn phasing into generic B-DNA. Therefore, if, as has been proposed, A-tracts have zero roll between the base-pairs and all curvature results from positive roll in the B-DNA segments, then this must be a general property of approximately 5 bp B-DNA sequences, not just special cases. This interpretation would require that the canonical structure of B-DNA be revised to include systematic roll between the base-pairs of about 6 degrees. Alternatively, the data are also consistent with zero average roll in the B-DNA sequences, and wedge angles dominated by negative roll in the A-tracts, or with an appropriate mixture of the two models. It is not possible to resolve this ambiguity using comparative electrophoresis or existing structural data. We show that published wedge angle parameters successfully predict the measured direction and, with appropriate rescaling, the magnitude of curvature due to a non-A-tract sequence containing the protein-free lac operator CAP protein binding site.

Base Sequence↗
PubMed1994

Mutagenesis supports water mediated recognition in the trp repressor-operator system.

High resolution crystallographic analysis of the trp repressor-operator complex indicates that the principal determinants of specificity are water mediated hydrogen bonds between the helix-turn-helix and the identity elements of the operator. One such hydration site involves a conserved G-C base pair (designated G6) six nucleotides away from the dyad which, if changed symmetrically to any other pair (e.g. G6-->A) reduces affinity to nonspecific levels. This same water site also contacts the conserved A5 which, if changed to G (mutation A5-->G), also diminishes affinity. The stereochemistry of the water mediated hydrogen bonding system predicts that the severe deterioration of in vitro binding caused by G6-->A should be reverted by a second deleterious mutation A5-->G. This proved to be the case. No other second mutation at conserved operator position 5 or 7 (flanking the G6-->A) reversed the effect of G6-->A.

Bacterial Proteins↗
PubMed1992

The DNA target of the trp repressor.

Unexpected features seen by high resolution X-ray crystallography at the interface of the trp repressor and the 'traditional' trp operator provoked the claim that the DNA fragment used in the crystal structure is not the true operator, and therefore that the crystal structure of the trp repressor-operator complex does not portray a specific interaction. An alternative sequence was proposed mainly on the basis of mutational studies and gel retardation analysis of short target duplexes (Staacke et al., 1990a,b). We have reexamined the sequence consensus in trpR-repressible promoters and analyzed the mutagenesis experiments of others including Staacke et al. (1990a) and found them fully consistent with the interactions of the traditional operator sequence seen in the crystal structure, and stereochemically inconsistent with the above referenced alternative model. Moreover, an in vitro trp repressor-DNA binding analysis, employing both novel DNA constructs devised to avoid previously encountered artifacts as well as full-length promoter sequences, indicates that the traditional operator used in the crystal structure is the preferred target of the trp repressor.

Bacterial Proteins↗
PubMed1989

Cooperativity in A-tract structure and bending properties of composite TnAn blocks.

The existence of intrinsically curved DNA molecules incorporating short runs of adenines is undisputed, but none of the current models can explain the entire experimental data set. Recently, Burkhoff and Tullius [Burkhoff, A. M., & Tullius, T. D. (1988) Nature 331, 455-457] offered an explanation for Hagerman's observations on A4T4N2 vs T4A4N2 polymers [Hagerman, P. J. (1986) Nature 321, 449-450], which showed that A4T4N2 multimers migrate anomalously slowly on polyacrylamide gels and T4A4N2 multimers migrate normally. In A4T4N2 multimers Burkhoff and Tullius observe a hydroxy-radical cutting pattern associated with bent DNA and a B-like cutting pattern in T4A4N2. They attribute this difference in cutting pattern to a clash in the TA step of T4A4N2 and suggest that TA4N5 might already adopt an unbent B-DNA conformation [Tullius T. D., & Burkhoff, A. M. (1988) in Structure and Expression. Vol. 3: DNA Bending and Curvature (Olson, W. K., Sarma, M. H., Sarma, R. H., & Sundaralingam, M., Eds.) pp 77-85, Adenine Press, Guilderland, NY]. We show that the conformation adopted by TnAn blocks is similar to that of AnTn blocks. Two A-tract structures of opposite polarity coexist in both blocks. Moreover, we demonstrate a cooperative buildup of a T-tract structure adjacent to an A-tract structure that cannot be predicted by any of the current models. We conclude that AA steps do not assume the same conformation in long tracts of A's as in isolated AA steps. Therefore, the assumption of nearest-neighbor models, that global curvature is an additive phenomenon of local effects, is invalid.

Adenine↗
PubMed1988

Low-temperature study of the A-DNA fragment d(GGGCGCCC).

The structure of the A-type duplex d(GGGCGCCC) was determined from data measured at 115 K to 2.0 A resolution. The space group, P4(3)2(1)2, is the same as for the 293 K structure; cell dimensions are a = 42.74 (4), c = 24.57 (1) A; R = 0.21 for 1694 observed reflections. The conformation and hydration are similar at the two temperatures. The average displacement parameters (B) for bases, sugars and phosphates all decrease by about 9 A2 relative to those found at 293 K. The individual values of B1/2 are linearly related to the distance from the molecular center of mass.

Base Sequence↗
PubMed1988

Phased psoralen cross-links do not bend the DNA double helix.

Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies base on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5 degrees [Pearlman, D. A., Holbrook, S. R., Pirkle, D. H., & Kim, S.-H. (1985) Science (Washington, D.C.) 227, 1304-1308]. On the other hand, Sinden and Hagerman [Sinden, R. R., & Hagerman, P. J. (1984) Biochemistry 23, 6299-6303] find that, in solution, psoralen cross-linked DNA is not bent. Here we use gel electrophoresis to test the validity of the current models. We have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28 degrees unwinding of the helix by HMT [Wiesehahn, G., & Hearst, J. E. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2703-2707], one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. We find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. Our observations place an upper limit of 10 degrees on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.

Cross-Linking Reagents↗
PubMed1987

The crystal structure of d(CCCCGGGG): a new A-form variant with an extended backbone conformation.

The crystal structure of d(CCCCGGGG) has been determined at a resolution of 2.25 A. The oligomers crystallize as A-DNA duplexes occupying crystallographic two-fold axes. The backbone conformation is, in general, similar to that observed in previously reported crystal structures of A-DNA fragments, except for the central linkage, where it adopts an extended structure resulting from all trans conformation at the P-O5'-C5'-C4' bonds. This type of conformation facilitates interstrand stacking between the guanines at the C-G site. The local helix twist at this step is very small (25 degrees) compared to an overall average of 33.5 degrees. The unique structure of the C-G base-pair step, namely the extended backbone and the distinct stacking geometry, may be an important feature in the recognition mechanism between double-stranded DNA molecules and restriction endonucleases such as Msp I, which cuts the sequence CCGG very specifically with a rate unaffected by neighboring base pairs.

DNA↗
PubMed1984

Base-stacking interactions in double-helical DNA structures: experiment versus theory.

Atom-atom potential energy calculations have been undertaken for deriving stacking energies in double-helical structures. A comparison between the energy patterns of A- and B-type double-helical fragments determined by single-crystal X-ray diffraction methods versus idealized uniform models based on fibre diffraction data shows that the van der Waals stacking energy is largely sensitive to local changes in the relative orientation of adjacent base pairs. The sequence-dependent conformational variability observed in the high-resolution structures appears to be a consequence of the equipartitioning of the stacking energy along the double helix. The large energy variations expected for a uniform structure are dampened considerably in the observed structures by means of local changes in conformational features such as helix rotation and roll angles between base pairs.

DNA↗