Rotation phosphodiester

Figure 3. Contour diagram of the base stacking potential energy Kg of sequential adenine bases and the hydrogen bonding potential energy VHB of the complementary A T base pairs as a function of the phosphodiester rotation angles

It has been proposed that the metal ion also has an interaction with the pro-Rp oxygen atom at the scissile phosphate, Pi.i. However, the scissile phosphodiester bond, within the crystal structure, is located approximately 20 A from the A9/G10.1 site [96]. It should be true that a conformationd change occurs to rotate the initial structure that is not feasible for the inhne attack to in-line structure around the scissile phosphate, but molecular dynamics analysis denies to arrange A9/G10.1 to come close to Pi.i via a metal ion. Furthermore, although it has been suggested that there is a weak interaction of a metal ion with the oxygen atom at P 1.1, our recent experiments observed no interaction of a metal ion with the Pi.i phosphate by kinetic and NMR analyses [97, 98]. 

The possible backbone phosphodiester conformations in a dinucleotide monophosphate and a dinucleotide triphosphate are investigated by semiempirical energy calculations. Conformational energies are computed as a function of the rotations o and <0 about the internucleotide P-0(3 ) and P-015 1 linkages, with the nucleotide residues themselves assumed to be in one of the preferred [C(3 )-e/K/o) conformations. 

The site of action of EcoRI is a specific hexanucleotide sequence. Two phosphodiester bonds are hydrolyzed (see arrows), resulting in fragmentation of both strands. Note the twofold rotational symmetry feature at the recognition site and the formation of cohesive ends. The weak base pairing between the cohesive ends is not sufficient to hold the two fragments together. 

SPECIFICITY. Restriction endonucleases are highly specific in hydrolyzing doubled-stranded DNA. Not only do they recognize a specific sequence of four to six base pairs (up to 11 base pairs in case of Bgl I see Table II) but the base pairs must be patind/iomic in the two strands of DNA. The cleavage sites possess twofold rotational symmetry as shown in Scheme I for restriction endonuclease Aat II from AcdtobacXdA OUddtL (Table II A, T, G, C indicate deoxyribonucleotides in this paper). In this example, the T-C phosphodiester bond in each strand two residues distal to the symmetry axis is cleaved by restriction endonuclease Aat II. 

Figure 111. Two idealized conformations of tfie Fiomo-DNA backbone. Conversion from A to B occurs through a 120° rotation about a and y of the phosphodiester linkage.

Structure of a phosphodiester group. The vertical arrows show the bonds in the phosphodiester group that are free to rotate. The horizontal arrows show the N-glycosidic bond about which the base can rotate freely. A polynucleotide consists of many nucleotides linked together by phosphodiester groups. 

Figure 1.61 Conformational freedom is heavily restricted in nucleic acids owing to lack of free rotation about 0 —P and P—0 bonds, (a) Nucleic acid equivalent of the Ramachandran plot illustrating the theoretically allowed angles of C and a. (b) Free rotation is primarily damped owing to the gauche effect in which lone-pair-o- orbital overlaps in phosphodiester links generate double bond character in 0—P bonds that restrict free rotation (adapted from Govil, 1976 [Wiley]).

Fig. 4 The proposed nucleotidyl transfer reaction mechanism. Step 1 The proton of the 3 OH of RNA primer transfers to a solvait water and a proton on water transfers to 02a of a-phosphate simultaneously Step 2 the proton of the 02a atom rotates to the side of P-phosphate Step 3 the 3 0 atom performs a nucleophilic attack at the a-phosphorus atom of the a-phosphate Step 4 the Pa-03p bond of the intermediate cleaves to form a phosphodiester bond and the proton on 02a migrates to 03 P...

The mechanism of hydrolysis is straightforward and resembles that for the hydroxide catalyzed hydrolysis of RNA with exception of specificity as only certain phosphodiester linkages are cleaved to give 3 - (and not 2 -) monophosphates (Fig. 3.7). However, with a knowledge of pseudo-rotation, two stereochemical mechanisms become possible for each step of the enzyme catalyzed reaction. 

Fig. 3.7. Differences between in-line and adjacent mechanism of phosphodiester bond hydrolysis, (a) First step (transesterification) in the RNase catalyzed hydrolysis of RNA illustrating two possible stereochemical pathways The in-line mechanism allows a direct displacement while the adjacent mechanism requires a pseudo-rotation, (b) Second step (hydrolysis) in the RNase catalyzed hydrolysis of RNA illustrating two possible stereochemical pathways. Again, the in-line mechanism allows a direct displacement to form the 3 -phosphate, while the adjacent mechanism requires a pseudorotation (26).

Various phosphodiester backbone substates can be found in X-ray crystal structmes of free nucleic acids and in complexes with proteins (Djuranovic and Hartmann 2004 Vamai et al. 2002). In particular, the coupled e (rotation around C3 -03 bond in C4 -C3 -03 -P) and ( (rotation around 03 -P bond in C3 -03 -P-05 ) in gauche-/trans and in trans/gauche-regimes, termed Bj and Bjj substates, respectively, are frequent in crystal structures of free DNA (Varnai et al. 2002). 

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