DNA Structure and Folding

Chromosomes, etc.

Let's look at how DNA is arranged in cells and viral particles. One of the problems facing organisms is how to pack the very long DNA molecule into the small volume of a cell.

Let's start with the simpler system of the T-even phages as represented by T2 in text Figure 24-1 (p 923) in the text. Note the huge amount of DNA which has been relaesed from the polyhedral head. For the related T4 phage the 168,889 bp DNA molecule is 60,800 nm in length and packs into a 210 nm long polyhedral head.

Similarly impressive packing is seen in E. coli which has a 1.7 mm long, 4,639,221 bp circular chromosome packaged into a "corner" of the 0.002 mm long bacterial cell. (micrograph, text Figure 24-4, p 926 and diagrammatically, text Figure 24-3, p 926-7)

Another even more complex packing is seen in eukaryote cells. (chromosome micrograph, text Figure 24-5a, p 927) As an example human cells have a total length of DNA per chromosome set (chromosome micrograph, text Figure 24-5b, p 927) of about 2 m (3.2 x 10⁹ bp), giving a total length of DNA in an adult of about 2 x 10¹⁴ m (0.02 light years).

So what's in all of this DNA? Let's look at Table 24-2 in the text (p 928). In particular we want to note the relationship of the number of "genes" vs. the amount of DNA.

Note that in going from E. coli and Homo sapiens the number of base pairs is increased by a factor of nearly 700 while the number of genes increases only by a factor of 7-8.

So what is different?

• In bacteria virtually all of the DNA is used as genes (it is translated into proteins or expressed as RNA machinery) and regulatory sequences. Most genes appear only once (some RNA's occur in small numbers of copies).
• In eukaryotes things are different.
  • Introns and Exons - we have looked at this aspect of eukaryote gene structure before as seen in text Figure 24-7 (p 929) there is commonly more DNA in introns which are not translated than in exon.
  • But there is much more DNA not involved in genes, and of uncertain usage, as shown in text Figure 24-8 (p 929)
    • Introns and Exons (seen above) approximately 5% of gene DNA is exon DNA.
    • Transposons (transposable elements which move around the genome) of three major types:
      • LINES: long interspersed elements (6-8 kbp each) often including genes coding for enzymes catalyzing transposition.
      • SINES: short interspersed elements (100-300 bp each) 2/3 are called Alu elements which usually include an AluI restriction recognition sequence.
      • Retrovirus-like sequences (1.5-11 kbp each) evolutionarily related to retroviruses like HIV.
    • SSR: simple-sequence repeats - usaully less than 10 bp each. Satellite DNA (centrifuge bands appear as satellites in CsCl centrifugation.
    • SD: large segmental duplications. These often appear multiple time in different locations.
    • A bunch of unknown purpose, though some of this encodes RNA.
  • Much of SSR DNA appears in Centromeres and Telomeres.
  • Telomers are needed in eukaryotes because can't readily replicate linear DNA ends, as we will see later.

The Supercoiling of DNA

DNA in vivo is supercoiled in various ways. Supercoiling allows DNA to take up a more compact form, and is a necessary consequence of replication and transcription.

So what is supercoiling? Supercoiling means that the helix is itself wrapped into a higher level coil, it is super-twisted or has super-helicity.

We will discuss supercoiling in terms of topology, the study of properties of geometric forms, and the transformations that leave them invarient in certain properties (e.g. a tea cup and a donut are topologically the same in that they have a single surface penetrated by a single hole).

Super helix topology. The topology of the DNA helix/supehelix can be described by a simple equation:

where:

• L = the linking number = the number of times one strand crosses the other. Note that this is invarient to twists and distortions so long as neither strand is cut! Thus if the primary helix is unwrapped, then it must wrap into a supercoil (demo with belt or tubing model).
• T = twist = number of complex revolutions one strand makes around the duplex axis in the conformation being considered.
  • T is positive (+) for right-handed duplex turns.
  • T is negative (-) for left-handed duplex turns.
  • For B-DNA the helix is right-handed, so T = + = (# base-pairs)/10.4, where 10.4 is the observed number of turns for aqueous B-DNA.
• W = writhing number = the number of turns the duplex axis makes around the superhelix axis in the conformation being considered. This is a measure of superhelicity.

So let's look at DNA in terms of topology. The best information is from viruses and plasmids - both are small enough to work with in whole form.

Bacteria, plasmids, mitochondria and viruses usually have circular DNA, or in the case of viruses, the DNA becomes circularized after injection into the host.

• Note the variable amounts of supercoiling in the micrographs of these viral genomes. (Figure 24-13, p 932)
• A twisted telephone cord, or a belt can demonstrate the differences between W (writhing) and T (twisting).
• For a closed circular piece of DNA note that the value of L is fixed, thus if the helix is overwound (a twist is added, T + 1), then it must supercoil in a negative sense (W must go negative by -1). In the example L = 9 and T changes from 9 to 10, thus forcing W to change from 0 to -1 (or vice versa). [overhead 28-36] (Figure 23-7, p 733)
  • Note that L is strictly only fixed for a closed circle, however, if the ends of a linear piece of DNA are fixed, as they effectively are by the bulk of a eukaryotic chromosome, then L will be effectively fixed for any relatively short sectin in the chromosomes interior.
• Experimentally the relationships of twist and writhing are observed in short closed circular genomes when the dye ethidium bromide (EtBr) is added incrementally. EtBr intercalates between bases in DNA, effectively forcing an unwinding of the helix (T decreases). Normal DNA is strongly negatively supercoiled, thus the addition of EtBr results in an incremental change of W from (-) to (+) as shown in the figure (inferred via centrifugation studies) [overhead 28-38] (Figure 24-13, p 932)

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Last modified 2 March 2009