Sandsmark, Fred, Contributing Writer has reference to this Academic Journal, PHwiki organized this Journal SEQUENCE SPECIFICITY DICTATED BY THE INTERPLAY BETWEEN DIRECT READOUT AND DNA FLEXIBILITY AT THE TATA BOX-BINDING PROTEIN – TATA BOX INTERFACE. Leonardo Pardo1, David Bosch1, Mercedes Campillo1, Nina Pastor2, in addition to Harel Weinstein3 1Unidad de Bioestadistica, Facultad de Medicina, Universidad Autonoma de Barcelona, 08193 Bellaterra, Spain; 2Facultad de Ciencias, UAEM, Av. Universidad 1001, Col. Chamilpa, 62210 Cuernavaca, Morelos, México; 3Department of Physiology in addition to Biophysics, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York NY 10029, U.S.A. ABSTRACT: A common mechanism of DNA bending by minor groove-binding proteins is the insertion of protein side chains between basepair steps, exemplified in TBP/DNA complexes. At the first in addition to last basepair steps of the TATA box, TBP kinks the DNA by inserting pairs of Phe side chains between the steps, in addition to placing Leu in addition to Pro side chains near the rim of the bases. QM calculations indicate that these side chains cannot discriminate between AT in addition to TA basepairs. The sequence selectivity is due to the differential DNA flexibility of the basepair steps, as revealed by MD/PMF calculations, in addition to to the ability of these steps to as long as m H-bonds in the major groove. At the central basepair step of the TATA box, TBP markedly untwists this step, while engaging in hydrogen bonds with the bases in addition to sugars. The H-bonds drive the con as long as mational transition at this step, but are not capable of discriminating between AA in addition to AT steps, as their strength is the same as long as both sequences. The calculated free energy cost as long as an equivalent con as long as mational transition is found to be sequence dependent, being higher as long as AA steps than as long as AT steps. Consequently, AA steps have a smaller distortion in TBP/DNA complexes than AT steps. ENERGETICS OF DIRECT READOUT SEQUENCE DEPENDENT DNA FLEXIBILITY 5’ KINK: KINK bps: TA, AT, TT, AA, CG TBP side chains: PHE, LEU AND PRO Forces: STACKING VAN DER WAALS CENTRAL BP STEP: CENTRAL bps: AA, AT TBP side chains: ASN, THR AND GLY Forces: H-BONDS 5’ KINK: Calculation of free energy differences as long as the B TA transition of various bps bps: F GTAT: 0.0 GATT: 2.3 GTTT: 4.7 GAAT: 6.9 CENTRAL BP STEP: Free energy calculation of the A TA (ATH VS. SCE) transition as long as TAAA VS. TATA FINDINGS The interaction of Phe side chains cannot discriminate among these four A T basepair steps, or between A T in addition to C G basepairs. Leu in addition to Pro side chains clash against the N2 amino group in C G basepairs, but cannot distinguish interactions with A T from T A basepairs. FINDINGS The strength of the H-bonds made from Asn in addition to Thr side chains to AA or AT basepair steps is practically the same. Complex ii compensates the poorer interaction with DNA by improving the interaction within TBP. Gly is important to stabilize the con as long as mation of the Asn in addition to Thr side chains. 1. DIRECT READOUT: Direct readout is not responsible as long as the selection of TA basepair steps at the 5’ kink. TBP tolerates equally well AA in addition to AT basepair steps at the central basepair step because the strength of the direct interactions to these two sequences is practically the same. ATH complex: AA step (Kim J.L et al., 1994) SCE complex: AT step (Kim Y. et al., 1993) -17.9 kcal/mol -17.8 kcal/mol -17.9 kcal/mol -17.9 kcal/mol -14.6 kcal/mol SCE complex (Kim Y. et al., 1993) Basepair F190 F207 P191 L205 ———- T2:A28 -4.4 -0.7 -1.6 A2:T28 -3.4 -0.7 -1.5 C2:G28 -3.0 -0.6 +1.5 ———- A3:T27 -7.0 -3.1 -1.6 T3:A27 -5.8 -3.5 -0.8 G3:C27 -7.1 -3.3 +32.9 TT/AA unfavorable due to steric clash in the major groove F(TA) < F(AT) due to better H-bonds From crystal structures in addition to calculations: AT is always more distorted than AA 2. DNA DISTORTION: 5’ KINK: TA steps are the easiest to bend into the TA-DNA con as long as mation, because of the interactions in the major groove in the final con as long as mation: two good intra-str in addition to H-bonds can be made in addition to there are no clashes. CENTRAL BASEPAIR STEP: AT steps are more distorted than AA steps in TBP-DNA complexes, because AT steps are easier to unwind in addition to bend than AA steps. transition F TATA SCE: 11.8 TAAA ATH: 8.1 INTRODUCTION: The TATA box-binding protein (TBP) binds specifically to 8 basepairs, using the minor groove surface of DNA. This mode of interaction is seen in all TBP-DNA complexes reported to date [1]. The TATA box consensus sequence is TATA@A@N, where @ is A or T. The minor groove of DNA is considered poor in in as long as mation content, given the very similar placement of H-bond acceptors (T-O2 in addition to A-N3) in addition to hydrophobic sites (A-C2) in A T in addition to T A basepairs. The TBP-TATA box interface is mostly hydrophobic, with Leu, Pro, in addition to Val side chains close to A-C2. H-bonds are only found at the central basepair step of the TATA element, between Asn in addition to Thr residues in addition to the H-bond acceptors in the minor groove. TBP bends in addition to untwists the TATA box drastically. There are 45º kinks at the first in addition to last basepair steps, in addition to a 20º unwinding at the central basepair step. The energetic cost of the various components of DNA distortions involved in the specific binding of TBP can be used to reveal the mechanisms underlying sequence specificity [2]. APPROACH: We use quantum mechanical calculations to examine the interactions between TBP side chains in addition to the basepair steps located at the most sequence conserved kink site (the 5’ kink: the first TA step, at the MP2 level), in addition to at the only step recognized through the as long as mation of H-bonds(the central basepair step, using DFT/BLYP3), to determine the role of direct readout in sequence discrimination. We use Molecular Dynamics/Potential of Mean Force calculations with the AMBER 4.1 potential 43to estimate the free energy cost of trans as long as ming B- in addition to A-DNA double str in addition to ed tetramers into the con as long as mations found in high resolution crystal structures of TBP-DNA complexes, to determine the role of DNA bendability in TATA box selection by TBP. Calculated F as long as fitting TAAA into the TATA structure of SCE: 14.4 - 11.8 = 2.6 kcal/mol CONCLUSIONS: REFERENCES: [1]. Kim, Y. et al. (1993) Nature 365:520; Kim, J.L. et al. (1994) Nature Struct. Biol. 1:638; Nikolov, D.B. et al (1996) PNAS, USA 93:4862; Ju, Z.S. et al. (1996) J. Mol. Biol. 261:239; Patikoglou, G.A. et al. (1999) Genes Dev. 13:3217 [2]. Pastor, N. et al. (1997) Biophys. J.73:640; Pastor, N. et al. (1997) in Molecular Modeling of Nucleic Acids (ACS, Leontis, N.B. in addition to SantaLucia Jr.,J., eds.) ) 268:329; Pardo, L., et al. (1998) Biophys. J. 74:2191; Pardo, L., et al. (2000) Biophys. J. in press; Pastor, N. in addition to Weinstein, H. (2000) in Theoretical Biochemistry (Elsevier, Eriksson, L. ed.) in press. Sanford-Brown Institute-Cranston RI

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