Principles of nucleic acid structure Biophysical Chemistry 1, Fall 2010 Reading assignment: Chap. 3 Web assignment: http://w3dna.rutgers.edu The phosphodiester linkage is directional. The 3´-oxygenNucleic acids:of nucleotide phosphates, i is joined sugars, to basesthe 5´-oxygen of nucleotide i+1. (i) (I+1) b541_Chapter-03.qxd 11/20/2008 10:56 AM Page 61 FA The sugar-phosphate backbone Basics of Nucleic Acid Structure 61 ζ O3’ α = O3’i-1—P—O5’—C5’ O P O α β = P—O5’—C5’—C4’ O5’ β Chain γ = O5’—C5’—C4’-C3’ nucleotide unit 5’ Base γ O4’ χ direction δ = C5’—C4’—C3’—C2’ 4’ 3’ 1’ δ ε = C4’—C3’—O3’—P ε 2’ i+1 O3’ ζ ζ = C3’—O3’—Pi+1-O3’i+1 P C3’ Residue i-1 O3’ OP1 P Residue i OP2 O5’ C5’ FIGURE 3.2 Section of the DNA backbone, showing the atom naming and the naming of var- ious torsion angles. The cyclic ring is the deoxyribose moiety of the backbone, and successive ribose units are connected via phosphate groups. The backbone is therefore often referred to as “the sugar-phosphate backbone.” Bottom: The direction of the chain is by convention defined by two oxygen atoms on the ribose, O5′ and O3′, and specifies the direction in which tran- scription takes place (from 5′ to 3′ in the new strand). main categories, E and T. If four atoms of the aldofuranose ring lie approxi- mately in a plane, the conformation is described as envelope (E), due to the pic- torial resemblance to an envelope, otherwise the conformation is described as twist (T) (Fig. 3.5). The puckering of the ribose is conveniently described by the phase angle P, ν ν which is defined in terms of the internal ribose conformation angles 0– 4 (see Fig. 3.6): (uu+-+ )( uu ) P = arctan 24 13 . 2up0[sin( 5 )+ sin( 25 p )] ° ° ν < = + ° P must be in the interval 0 –360 , so if 0 0 then P P 180 . The various con- formational nomenclatures of the ribose ring can conveniently be drawn along a circle as a function of P (Fig. 3.7). Adenine and guanine are the two most common purine bases, but inosine is found in some nucleic acid molecules. DNA has two types of pyrimidines, cyto- sine and thymine. In RNA, thymine is normally replaced by a similar base, uracil, which lacks the methyl group found in thymine (Fig. 3.8). b541_Chapter-03.qxd 11/20/2008 10:56 AM Page 63 FA Sugar puckering Basics of Nucleic Acid Structure 63 2’ 3’ C5’ B 2E B C5’ 3E 3’ O4’ O4’ 2’ C5’ 2’ 3’ B 2 B C5’ 3 T3 T2 3’ O4’ 2’ B C5’ O4’ 2’ B C5’ 3’ O4’ E3 E2 3’ 2’ C5’ B B C5’ S conformations N conformations FIGURE 3.5 Various sugar ring puckering conformations. Those on the left are denoted S (for south); those on the right, N (for north). The C3′-endo conformation is seen at the top right, and the C2′-endo conformation at the top left. The notation of E and T conformations is also given. Superscript numbers preceding E or T refer to carbon atoms on the same side of the reference plane (horizontal line) as C5′. Subscripts following E or T denote atoms on the opposite side of the reference plane. ν4 O4’ ν0 ν0 = C4’—O4’—C1’—C2’ ν1 C4’ C1’ = O4’—C1’—C2’—C3’ ν2 ν3 ν1 = C1’—C2’—C3’—C4’ C3’ C2’ ν3 = C2’—C3’—C4’—O4’ ν2 ν4 = C3’—C4’—O4’—C1’ FIGURE 3.6 Naming scheme for the torsion angles of the sugar ring in nucleotides. There are two preferred ways of arranging the base in relation to the ribose, syn and anti (Figs. 3.9 and 3.10). In pyrimidine nucleotides only the anti confor- mation is found, because this avoids collision between the oxygen and the ribose. Purines can have both orientations, but the anti conformation is the most common of them. Mammalian DNA is known to contain a methylated base, 5-methylcytosine (m5C). Bacterial DNA contains this one and two other methylated bases, namely, b541_Chapter-03.qxd 11/20/2008 10:56 AM Page 64 FA Sugar puckering 64 A Textbook of Structural Biology N C2’-exo0˚ C3’-endo 324˚ 36˚ E 3T 3 C1’-endo 1 2 2 E 3 C4’-exo 2T 4T 1 E 4E 288˚ 1 0 72˚ 0T 4T 0 O4’-exo 0E E C4’-endo 4 0 0T 1T 252˚ 108˚ 4 E 1E 4 2 C4’-endo T T C1’-exo 3 E 2 2 1 3 3T E 216˚ 144˚ C3’-exo 180˚ C2’-endo S FIGURE 3.7 Diagram showing the correlation between the phase angle P and the ribose con- formation. Conformational angles of P are divided into two categories, north (N) and south (S). NH2 O N 6 N 6 7 5 1 N 7 5 1NH 8 8 9 4 2 9 4 2 NH 3 NH 3 N N NH2 Adenine Guanine O O NH2 4 4 4 5 3 NH 5 3 NH 5 3 N 6 2 6 2 6 2 1 1 NH O NH O NH1 O Uracil Thymine Cytosine FIGURE 3.8 The most common bases found in nucleic acids: the top row is purines; the bottom row pyrimidines. The atom-numbering scheme of purines and pyrimidines is given. b541_Chapter-03.qxd 11/20/2008 10:56 AM Page 64 FA 64 A Textbook of Structural Biology N C2’-exo0˚ C3’-endo 324˚ 36˚ E 3T 3 C1’-endo 1 2 2 E 3 C4’-exo 2T 4T 1 E 4E 288˚ 1 0 72˚ 0T 4T 0 O4’-exo 0E E C4’-endo 4 0 0T 1T 252˚ 108˚ 4 E 1E 4 2 C4’-endo T T C1’-exo 3 E 2 2 1 3 3T E 216˚ 144˚ C3’-exo 180˚ C2’-endo S FIGURE 3.7 Diagram showing the correlation between the phase angle P and the ribose con- formation. Conformational angles of P are divided into two categories, north (N) and south (S). purines and pyrimidines NH2 O N 6 N 6 7 5 1 N 7 5 1NH 8 8 9 4 2 9 4 2 NH 3 NH 3 N N NH2 Adenine Guanine O O NH2 4 4 4 5 3 NH 5 3 NH 5 3 N 6 2 6 2 6 2 1 1 NH O NH O NH1 O Uracil Thymine Cytosine FIGURE 3.8 The most common bases found in nucleic acids: the top row is purines; the bottom row pyrimidines. The atom-numbering scheme of purines and pyrimidines is given. b541_Chapter-03.qxd 11/20/2008 10:56 AM Page 65 FA The glycosidic torsion parameter Basics of Nucleic Acid Structure 65 FIGURE 3.9 The anti and syn conformations of adenine monophosphate. 0˚ 0˚ syn 4’ 4’ 60˚ O4’ 300˚ 60˚ O4’ 300˚ χ -60˚ -60˚ 3’ 3’ C4 Purine Pyrimidine 2’ 2’ χ C2 -120˚ O 120˚ 240˚ 120˚ 240˚ anti 180˚ 180˚ FIGURE 3.10 Diagram defining the torsion angle χ around the N-glycosidic bond. The penta- gon illustrates the ribose unit, and the base is seen edge-on. The sequence of atoms chosen to define this angle is O4′–C1′–N9–C4 for purine and O4′–C1′–N1–C2 for pyrimidine deriva- tives. Thus when χ = 0° the O4′–C1′ bond is eclipsed with the N9–C4 bond for purine and the N1–C2 bond for pyrimidine derivatives. The syn conformation is defined as χ =±90° and anti as χ = 180 ± 90°; thus, the syn conformational region is given by the upper half-circle, and the anti conformation by the lower half. The purine on the left is therefore in the syn conformation, and the pyrimidine on the right, in the anti conformation. b541_Chapter-03.qxd 11/20/2008 10:56 AM Page 68 FA Watson-Crick base pairing 68 A Textbook of Structural Biology major groove major groove H N O H N H H O N N N NH N N HN R N N N N R N O N H O R R H minor groove minor groove Guanine Cytosine Adenine Thymine FIGURE 3.13 The Watson–Crick base pairs. The sugar moieties are represented by R. Notice that the GC base pair on the left interacts via three hydrogen bonds, whereas the AT base pair on the right has only two. This makes the GC base pair and thus GC-rich DNA more stable than the AT base pair and AT-rich DNA. phosphodiester linkages between adjacent nucleotides. In the sugar-phosphate part the phosphate groups connect to the 3′ carbon of one deoxyribose moiety and the 5′ carbon of the next moiety, thereby linking successive deoxyriboses together. The two ends of a chain differ; the end where the 5′ carbon is not con- nected to another nucleotide is called the 5′ end. The other end is called the 3′ end. The two ends may have or lack free phosphate groups. Each of the four bases in DNA has a unique set of hydrogen bond donors and acceptors that allows it to form base pairs with the other bases. In double-stranded DNA we have AT (adenine-thymine) base pairs with two hydrogen bonds and GC (guanine-cytidine) base pairs with three hydrogen bonds (Fig. 3.13). These inter- actions are called Watson–Crick base pairs to honor the scientists who first sug- gested that these base pairs are the basis of heredity.