Chapter 28: Biomolecules: Nucleic Acids
Base, sugar, phosphate — and the two names for two of them
A nucleic acid looks intimidating until you take one residue apart, and then it is three familiar pieces bolted together. Reading from the outside in: a nitrogen-containing aromatic heterocycle, the base; a five-membered-ring sugar, always a furanose; and a phosphate group esterifying one of the sugar's hydroxyls. Every piece is chemistry you have already met — an aromatic heterocycle from the amines chapter, a cyclic hemiacetal from the carbohydrates chapter, and a phosphate ester that behaves much like the carboxylic esters of chapter 21. Two of these assemblies have names worth keeping…
Two ring systems, five bases, and the lactam form that does the work
Only five heterocycles do the work of storing genetic information, and they are built from just two ring systems. The purines, adenine and guanine, are bicyclic: a six-membered pyrimidine ring fused along an edge to a five-membered imidazole ring, giving a flat nine-atom framework numbered N1 through C6 around the six-membered ring and N7, C8, N9 around the five-membered one. The pyrimidines, cytosine, thymine, and uracil, are the single six-membered ring with nitrogens at positions 1 and 3. It is worth noticing that the numbering runs in opposite senses in the two systems, which is a…
A beta-N-glycoside at the anomeric carbon
Joining the base to the sugar uses chemistry already established in the carbohydrate chapter, with nitrogen in place of oxygen. The bond runs from a nitrogen of the base to C1' of the furanose — the anomeric carbon, the one that was the aldehyde carbon before the open-chain sugar closed its ring. Which nitrogen is used is not a free choice. A purine attaches through N9, the nitrogen of the five-membered imidazole ring that carries the hydrogen in the free base. A pyrimidine attaches through N1. Any other attachment gives a compound that is not a natural nucleoside, and the choice is not…
One phosphate, two esters, and a strand with a direction
The polymer bond in a nucleic acid is a phosphate ester, and a single phosphorus atom does the joining. Phosphoric acid has three hydroxyl groups. In the backbone, two of them are esterified — one to the 3'-oxygen of one sugar and one to the 5'-oxygen of the next — which makes each linkage a phosphodiester. Three things about that description are worth stating explicitly, because each is a common place to go wrong. The 2' position is never used in the backbone, even in RNA where it is available. The bases take no part in the connection at all; they hang off the chain rather than forming it,…
Reading donors and acceptors off the structures
The specificity of base pairing looks like something to memorize and is in fact something to read off the structures. Two independent conditions have to be met at once, and keeping them separate in your mind is what makes the rules derivable rather than arbitrary. The first condition is geometric. The two sugar-phosphate strands of a duplex run at a fixed separation, so every rung of the ladder must span the same distance — if some rungs were longer than others the backbone would have to buckle. A purine, being a fused bicycle, is roughly twice the width of a pyrimidine. Two purines facing…
Antiparallel strands, two grooves, and the role of stacking
The duplex is the arrangement that satisfies every constraint the preceding sections established, simultaneously. In the B form, the one that dominates for hydrated DNA under physiological conditions, two strands wind around a shared axis in a right-handed helix. The polyanionic backbones sit on the outside, in contact with water and counterions, and the flat, hydrophobic base pairs stack in the interior roughly perpendicular to the helix axis, about ten and a half pairs per turn, 3.4 Å apart along the axis, in a helix about 20 Å across. The arrangement is exactly what you would predict for…
A nucleophile tethered two atoms from its target
Here is an experiment with a striking result. Take a short RNA and a DNA of matching sequence, and treat both with dilute aqueous sodium hydroxide at room temperature. Within minutes the RNA has been chopped into small fragments. The DNA is still intact days later. Any explanation has to account for a rate difference of many orders of magnitude between two molecules that look almost the same. The backbone is the same 3'-to-5' phosphodiester in both. The bases differ only in thymine against uracil, which is not near the reacting center. The single structural difference that could matter is the…
The 3'-hydroxyl attacks the alpha phosphorus
Replication and transcription look like different biological processes and are, at the level of chemistry, the same reaction repeated. Learning it once covers both, and covers the reverse transcription of retroviruses as well. Start by inventorying what is present at the growing point. The growing strand ends in a free 3'-hydroxyl. The incoming monomer is a nucleoside 5'-triphosphate — dATP, dGTP, dCTP, or dTTP for DNA; ATP, GTP, CTP, or UTP for RNA — carrying three phosphorus atoms in a row, labeled alpha, beta, and gamma counting outward from the sugar. A template strand is present, holding…
Designing a substrate that cannot be extended
The chain-extension mechanism has an obvious point of attack, and medicinal chemistry has exploited it about as thoroughly as any mechanism in biology. Extension requires a free 3'-hydroxyl on the residue that was added last. If a residue is incorporated that does not have one, the chain cannot continue: there is no nucleophile available for the next substitution at phosphorus. This is the chain terminator strategy, and what makes it elegant is that the drug is not an inhibitor in the ordinary sense at all. It does not block the active site or compete for it in a way the enzyme can shrug off.…
Sanger sequencing and the phosphoramidite cycle
The two laboratory operations on DNA — reading it and writing it — use quite different chemistry, and both follow directly from principles already established. Neither is a new mechanism; they are applications. Reading: Sanger sequencing. Set up an ordinary polymerase extension from a primer annealed to the template, with all four normal deoxynucleoside triphosphates present, and add a small proportion of dideoxynucleoside triphosphates — the 2',3'-dideoxy analogs from the previous section. A dideoxy residue is incorporated by the polymerase exactly as a normal one would be, since it is…
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