Nucleotides: Structure and DNA vs RNA

Nucleotides are the building blocks of nucleic acids, each consisting of a nitrogenous base, a pentose sugar and one or more phosphate groups; DNA (with deoxyribose and the bases A, T, G, C) stores genetic information in a double helix, and RNA (with ribose and A, U, G, C) is usually single-stranded and carries out the use of that information. In brief: in DNA, adenine pairs with thymine through two hydrogen bonds and guanine pairs with cytosine through three, and the haploid human genome of 3.1 billion base pairs would be about 1 m long if stretched out.

The sequence of nucleotides in DNA contains the instructions for making every protein in an organism, and it is copied faithfully from cell to cell and from parent to offspring. Nucleotides also serve as energy carriers (ATP) and signaling molecules. This article describes the structure of a nucleotide, compares DNA and RNA in a table, explains base pairing and the double helix, and shows calculations of genome length, mass and melting temperature, with a short history of the discovery of the structure of DNA.

What is a nucleotide?

A nucleotide has three parts:

• a pentose sugar: ribose in RNA, 2′-deoxyribose in DNA (which lacks the –OH on carbon 2′);
• a nitrogenous base attached to carbon 1′ of the sugar by a glycosidic bond;
• one to three phosphate groups attached to carbon 5′.

The combination of base and sugar without the phosphate is a nucleoside (adenosine, guanosine, cytidine, thymidine, uridine). The numbers of the sugar carbons carry a prime (1′ to 5′) to distinguish them from the numbering of the atoms in the base. Nucleotides with three phosphates, ATP, GTP, CTP, UTP and the deoxy forms, are the starting materials for the synthesis of nucleic acids.

Which bases are there?

• Purines (two fused rings): adenine (A) and guanine (G).
• Pyrimidines (one ring): cytosine (C), thymine (T, in DNA only) and uracil (U, in RNA only). Thymine is 5-methyluracil.

A mnemonic: “pure silver” for pyrimidines (cytosine, thymine, uracil: C, T, U) and “PURe As Gold” for the purines (A, G).

How are nucleotides joined?

The nucleotides are linked by phosphodiester bonds: the phosphate on the 5′ carbon of one nucleotide forms an ester with the 3′ hydroxyl of the next, with loss of water (pyrophosphate in the biological synthesis). This gives a sugar–phosphate backbone with the bases sticking out. The chain has a direction: one end has a free 5′ phosphate (the 5′ end), and the other a free 3′ hydroxyl (the 3′ end). By convention, a sequence is written from 5′ to 3′: 5′-ATGC-3′. Each phosphate has a negative charge at physiological pH, so nucleic acids are polyanions, hence acids.

What is the structure of DNA?

DNA consists of two polynucleotide strands wound around each other in a right-handed double helix. Their main features are:

• Antiparallel strands: one runs 5′→3′, the other 3′→5′.
• Base pairing: adenine pairs with thymine (two hydrogen bonds), and guanine with cytosine (three hydrogen bonds). A purine always pairs with a pyrimidine, so that the width of the helix is constant (about 2.0 nm). The sequence of one strand determines the other (complementary strand), which is the basis for replication.
• Chargaff’s rules: in double-stranded DNA the amount of A equals T, and G equals C. For human DNA, the composition is about 30 % A, 30 % T, 20 % G and 20 % C.
• Dimensions of B-DNA: 0.34 nm between adjacent base pairs, 10.5 base pairs per turn, so one turn is 3.4–3.6 nm long. The sugar–phosphate backbones are outside, the bases are inside and are stacked on each other, which gives most of the stability (stacking interactions and the hydrophobic effect), in addition to the hydrogen bonds.
• Grooves: the helix has a wide major groove and a narrow minor groove, where proteins recognize specific sequences.

What is RNA?

RNA differs from DNA in having ribose, uracil instead of thymine, and usually in being single-stranded, which allows it to fold into complex shapes by base pairing within the same strand (hairpins, loops). The extra 2′–OH group makes RNA more reactive and less stable than DNA, which is suitable for a temporary message. The main types are:

• Messenger RNA (mRNA): carries the code of a gene from DNA to the ribosome.
• Transfer RNA (tRNA): brings amino acids to the ribosome; it has about 76 nucleotides and a cloverleaf shape.
• Ribosomal RNA (rRNA): makes up most of the mass of the ribosome and catalyzes the formation of peptide bonds.
• Others: microRNAs and small interfering RNAs that regulate genes, and catalytic RNAs (ribozymes).

DNA and RNA compared

Worked examples

Example 1: the complementary strand

The strand 5′-ATGCCGTA-3′ has the complementary strand 3′-TACGGCAT-5′ (A–T, T–A, G–C, C–G, and so on). Read in the standard 5′→3′ direction, the complementary strand is 5′-TACGGCAT-3′, which is the “reverse complement” of the first strand.

Example 2: base composition

A sample of double-stranded DNA has 22 % guanine. By Chargaff’s rule, cytosine is also 22 %, so G + C = 44 %. The remaining 56 % are A and T, 28 % each.

Example 3: the length of the human genome

The haploid human genome has about 3.1 × 10⁹ base pairs. The length is 3.1 × 10⁹ × 0.34 nm = 1.05 × 10⁹ nm = 1.05 m. A diploid cell with two copies has about 2 m of DNA in a nucleus with a diameter of 6 µm, a compaction by a factor of about 3 × 10⁵ in length, achieved by wrapping around histone proteins into chromatin. With about 3.7 × 10¹³ cells in a human body, the total length of DNA is about 7 × 10¹³ m, which is about 470 times the distance from the Earth to the Sun.

Example 4: the mass of the genome

The average mass of a base pair is about 650 g/mol. For 3.1 × 10⁹ bp the molar mass is 2.0 × 10¹² g/mol, and the mass of one haploid genome is 2.0 × 10¹² / 6.022 × 10²³ = 3.3 × 10⁻¹² g = 3.3 pg. A diploid cell has about 6.6 pg of nuclear DNA.

Example 5: the melting temperature

Heating separates the two strands (denaturation or “melting”). The temperature at which half of the DNA is single-stranded depends on the proportion of G–C pairs, because each has three hydrogen bonds and more stacking. For long DNA in a typical salt solution, T_m ≈ 69.3 + 0.41 × (%GC) °C. For 40 % GC: T_m = 69.3 + 16.4 = 85.7 °C. For 60 % GC: T_m = 69.3 + 24.6 = 93.9 °C. This is the basis of the heating step of PCR.

Example 6: the number of hydrogen bonds

A DNA fragment of 20 base pairs with 12 A–T and 8 G–C pairs has 12 × 2 + 8 × 3 = 48 hydrogen bonds.

How was the structure of DNA discovered?

Friedrich Miescher isolated “nuclein” from the nuclei of white blood cells in 1869. Phoebus Levene identified the components in the early 20th century, ribose in RNA in 1909 and deoxyribose in DNA in 1929, but wrongly thought that the molecule was a short repetitive tetranucleotide. Erwin Chargaff showed in 1950 that the proportions of the bases in DNA vary between species, but that A = T and G = C. Oswald Avery, Colin MacLeod and Maclyn McCarty showed in 1944 that DNA, and not protein, carries genetic information. Rosalind Franklin and Maurice Wilkins obtained X-ray diffraction patterns of DNA fibers, and in 1953 James Watson and Francis Crick proposed the double helix on the basis of this evidence (“Photograph 51”) and of Chargaff’s rules. Watson, Crick and Wilkins received the Nobel Prize in Physiology or Medicine in 1962; Franklin died in 1958.

Which mistakes are common?

• Mixing up the bases of DNA and RNA: thymine in DNA, uracil in RNA.
• Pairing A with G: purines pair with pyrimidines: A–T and G–C.
• Forgetting that the strands are antiparallel: read the complementary strand in the reverse direction.
• Confusing nucleoside and nucleotide: a nucleotide has a phosphate.
• Counting the hydrogen bonds wrongly: A–T has two, G–C three.