Module 7 : Microbial Molecular Biology and Genetics

Lecture 1 : Structure and Function of Genetic Material

In living organisms DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together. These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a nucleobase, which interacts with the other DNA strand in the helix. A nucleobase linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. Polymers comprising multiple linked nucleotides (as in DNA) are called a polynucleotide.

The backbone of the DNA strand is made from alternating phosphate and sugar residues. The sugar in DNA is 2-deoxyribose, which is a pentose (five- carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel . The asymmetric ends of DNA strands are called the 5′ (five prime) and 3′ (three prime) ends, with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.

The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among the aromatic nucleobases. In the aqueous environment of the cell, the conjugated π bonds of nucleotide bases align perpendicular to the axis of the DNA molecule, minimizing their interaction with the solvation shell and therefore, the Gibbs free energy. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar/phosphate to form the complete nucleotide.

Fig. 5 . Chemical structure of DNA, with colored label identifying the four bases as well as the phosphate and deoxyribose components of the backbone.

The nucleobases are classified into two types: the purines, A and G, being fused five- and six-membered heterocyclic compounds, and the pyrimidines, the six-membered rings C and T. A fifth pyrimidine nucleobase, uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. Uracil is not usually found in DNA, occurring only as a breakdown product of cytosine. In addition to RNA and DNA a large number of artificial nucleic acid analogues have also been created to study the proprieties of nucleic acids, or for use in biotechnology.

Twin helical strands form the DNA backbone. Another double helix may be found by tracing the spaces, or grooves, between the strands. Grooves are adjacent to the base pairs and may provide a binding site. As the strands are not directly opposite each other, the grooves are unequally sized. One groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide. The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove.

In a DNA double helix, each type of nucleobase on one strand normally interacts with just one type of nucleobase on the other strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with A bonding only to T, and C bonding only to G. This arrangement of two nucleotides binding together across the double helix is called a base pair. As hydrogen bonds are not covalent , they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can therefore be pulled apart like a zipper, either by a mechanical force or high temperature. As a result of this complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. Indeed, this reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in living organisms.

The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds. DNA with high GC-content is more stable than DNA with low GC-content.

As noted above, most DNA molecules are actually two polymer strands, bound together in a helical fashion by noncovalent bonds; this double stranded structure (dsDNA) is maintained largely by the intrastrand base stacking interactions, which are strongest for G, C stacks. The two strands can come apart – a process known as melting – to form two ss DNA molecules. Melting occurs when conditions favor ssDNA; such conditions are high temperature, low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH is rarely used).