Gene organization and expression in eukaryotes

Publisher Summary

This chapter discusses gene organization and expression in eukaryotes. There are a number of fundamental differences between the way genes are arranged, expressed, and controlled in eukaryotic cells when compared with bacteria. Eukaryotes, because of their greater complexity, must possess much more genetic information. Primary RNA transcripts in eukaryotic cells are extensively modified, cleaved, and spliced in the nucleus before being transported to the cytoplasm in the form of mRNA. The control of gene expression is much more complex and diverse in eukaryotes, with many levels of regulation. In addition to transcriptional control, posttranscriptional mechanisms also play a major role. The primary translation products may, in turn, be subjected to posttranslational modification. DNA replication in eukaryotic cells is fundamentally the same as that in bacteria. Replication is semi-conservative, occurs in a 5’ → 3’ direction, and requires a template, a primer, and deoxyribonucleoside triphosphates for synthesis to occur. Replication also precedes bidirectionally, but in view of the large size of eukaryotic chromosomes, synthesis is initiated simultaneously at many origins; otherwise chromosome replication would take many days.

Genes and chromosomes

There are a number of fundamental differences between the way genes are arranged, expressed and controlled in eukaryotic cells, when compared with bacteria.

1. Eukaryotes, because of their greater complexity, must possess much more genetic information. For example, a human cell contains about 1000 times more DNA than an E. coli cell. As a result the DNA may have an overall length of several centimetres which must be packed into a few micrometres. Hence the DNA of eukaryotic cells is very highly condensed and this is aided by the presence of a class of basic proteins called histories. Bacterial DNA is not associated with histones.

2. Eukaryotic chromosomes are located within a nucleus bounded by a nuclear membrane and, since proteins are synthesized in the cytoplasm, the sites of transcription and translation are physically separated. Consequently these two processes are not as closely coupled as they are in bacteria.

3. Primary RNA transcripts in eukaryotic cells are extensively modified, cleaved and spliced in the nucleus before being transported to the cytoplasm in the form of mRNA.

4. The control of gene expression is much more complex and diverse in eukaryotes, with many levels of regulation. In addition to transcriptional control, post-transcriptional mechanisms also play a major role. Furthermore, the primary translation products may in turn be subjected to post-translational modification.

Eukaryotic chromosome structure

It was not known for many years whether each eukaryotic chromosome contained a single linear DNA duplex molecule. Very large DNA molecules are readily broken down during isolation and handling, due to the shearing forces involved when working with molecules several millimetres or more in length. Eventually this problem was overcome by using a sophisicated viscoelastic technique which involved stretching the DNA molecules out in a flow of liquid, and then allowing them to recoil to their normal state. A measure of the rate of recoiling provides a measure of the molecular weight of the largest molecules in the preparation. When cells from fruit flies were lysed and subjected to this technique the mass of the largest DNA molecule was found to be 41 × 10⁹ daltons; a value which agreed well with that for the DNA content of the largest fruit fly chromosome. Subsequent autoradiographic studies of radioactively labelled DNA confirmed the existence of very long DNA molecules and showed them to be linear and unbranched.

As mentioned above, the DNA in eukaryotic chromosomes is tightly bound to a group of small basic proteins called histones. The protein: DNA ratio is about 1:1 and the complex is known as chromatin. Histones can be fractionated into five types called Hl, H2A, H2B, H3 and H4. They are all extremely basic, about one-quarter of their residues being either lysine or arginine. Each type of histone can be found in a variety of forms due to varying levels of post-translational modification of some of the amino acid side chains. These include acetylation, methylation and phosphorylation, and may be important in regulating DNA replication and transcription although this has yet to be firmly established. Histones H3 and H4 are highly conserved between species. In fact calf and pea H4 histones only differ by two conservative changes in their amino acid sequence, indicating that these histones play a crucial role in chromatin structure that was established early in the evolution of eukaryotes and has hardly diverged since.

Nucleosomes

In 1974, Roger Kornberg proposed that chromatin is composed of regularly repeating units which he called nucleosomes. When observed in the electron microscope adjacent nucleosomes were joined together giving the appearance of beads on a string.

Several lines of evidence have shown that each nucleosome is composed of a core particle containing 140 base pairs of DNA (Figure 21.1), irrespective of the species or cell type. This DNA is wound in the form of 1·75 turns of a left-handed superhelix, round a histone octamer composed of two molecules each of H2A, H2B, H3 and H4. Adjacent nucleosomes are then joined by a short stretch of DNA, called linker DNA, thereby maintaining the integrity of the chromosome. Consequently a typical mammalian gene of 10000 base pairs will contain about 40 nucleosomes. Histone H1, which is present at only one molecule per nucleosome and is absent from the core particle, may be associated with the linker DNA and serve as a bridge between adjacent nucleosomes.