GENOME ORGANISATION IN PROKARYOTES,

EUKARYOTES AND VIRUSES

MOLECULAR BIOLOGY

AMAN KHAN| BSC. (H) MICROBIOLOGY 3RD SEM| 4607

ORGANISATION OF EUKARYOTIC GENOME:
A sequence of four nucleotides arranged in a certain pattern, encoding information, constitutes an organism’s
genetic material or DNA (deoxyribonucleic acid). The linear arrangement of DNA components and their partition
into chromosomes is referred to as a genomic organisation. “Genome organisation” can also refer to the arrangement
of DNA sequences inside the nucleus and the three-dimensional structure of chromosomes.

Eukaryotic genomes are linear and follow the Watson-Crick Double Helix structural model. They are contained
within chromosomes, bundles of DNA and proteins (Histone) known as nucleosomes. The protein-coding genes in
eukaryotic genomes are organised in exons and introns, which represent the coding sequence and intervening
sequence, respectively, indicating the functionality of the RNA section of the genome.

DNA, Histones, and Chromatin

Certain proteins compact chromosomal DNA into the microscopic space of the eukaryotic nucleus. These proteins
are called histones, and the resulting DNA-protein complex is called chromatin. It may seem paradoxical that
proteins are added to DNA to make it more compact. However, if you have ever tried to store a garden hose, you
know that it is much easier to do so if you begin by coiling the hose. Of course, coiling requires work, and energy is
needed to perform work. Thus, within the nucleus, histones provide the energy (mainly in the form of electrostatic
interactions) to fold DNA. As a result, chromatin can be packaged into a much smaller volume than DNA alone.

Histones are a family of small, positively charged proteins termed H1, H2A, H2B, H3, and H4. DNA is negatively
charged, due to the phosphate groups in its phosphate-sugar backbone, so histones bind with DNA very tightly.

Chromosomal DNA is packaged inside microscopic nuclei with the help of histones. These are positively-charged
proteins that strongly adhere to negatively-charged DNA and form complexes called nucleosomes. Each nuclesome
is composed of DNA wound 1.65 times around eight histone proteins. Nucleosomes fold up to form a 30-nanometer
chromatin fiber, which forms loops averaging 300 nanometers in length. The 300 nm fibers are compressed and
folded to produce a 250 nm-wide fiber, which is tightly coiled into the chromatid of a chromosome.

The Nucleosome: The Unit of Chromatin

The basic repeating structural (and functional) unit of chromatin is the nucleosome, which contains eight histone
proteins and about 146 base pairs of DNA. The observation by electron microscopists that chromatin appeared
similar to beads on a string provided an early clue that nucleosomes exist. Another clue came from chemically cross-
linking (i.e., joining) histones in chromatin. This experiment demonstrated that H2A, H2B, H3, and H4 form a
discrete protein octamer, which is fully consistent with the presence of a repeating histone-containing unit in the
chromatin fiber.

Today, researchers know that nucleosomes are structured as follows: Two each of the histones H2A, H2B, H3, and
H4 come together to form a histone octamer, which binds and wraps approximately 1.7 turns of DNA, or about 146
base pairs. The addition of one H1 protein wraps another 20 base pairs, resulting in two full turns around the
octamer, and forming a structure called a chromatosome. The resulting 166 base pairs is not very long, considering
that each chromosome contains over 100 million base pairs of DNA on average. Therefore, every chromosome
contains hundreds of thousands of nucleosomes, and these nucleosomes are joined by the DNA that runs between
them (an average of about 20 base pairs). This joining DNA is referred to as linker DNA. Each chromosome is thus
a long chain of nucleosomes, which gives the appearance of a string of beads when viewed using an electron
microscope.

The amount of DNA per nucleosome was determined by treating chromatin with an enzyme that cuts DNA (such
enzymes are called DNases). One such enzyme, micrococcal nuclease (MNase), has the important property of
preferentially cutting the linker DNA between nucleosomes well before it cuts the DNA that is wrapped around
octamers. By regulating the amount of cutting that occurs after application of MNase, it is possible to stop the
reaction before every linker DNA has been cleaved. At this point, the treated chromatin will consist of
mononucleosomes, dinucleosomes (connected by linker DNA), trinucleosomes, and so forth. If DNA from MNase-
treated chromatin is then separated on a gel, a number of bands will appear, each having a length that is a multiple of
mononucleosomal DNA. The simplest explanation for this observation is that chromatin possesses a fundamental
repeating structure. When this was considered together with data from electron microscopy and chemical cross-
linking of histones, the "subunit theory" of chromatin was adopted. The subunits were later named nucleosomes and
were eventually crystallized. The model of the nucleosome that crystallographers constructed from their data is
shown in Figure 3. Phosphodiester backbones of the DNA double helix are shown in brown and turquoise, while
histone proteins are shown in blue (H3), green (H4), yellow (H2A), and red (H2B). Note that only eukaryotes (i.e.,
organisms with a nucleus and nuclear envelope) have nucleosomes. Prokaryotes, such as bacteria, do not.
 Fig. Electron micrograph of chromatin: the beads on a string.

Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones (brown and turquoise) and eight
histone protein main chains (blue: H3; green: H4; yellow: H2A; red: H2B)

Chromatin Is Coiled into Higher-Order Structures

The packaging of DNA into nucleosomes shortens the fiber length about sevenfold. In other words, a piece of DNA
that is 1 meter long will become a "string-of-beads" chromatin fiber just 14 centimeters (about 6 inches) long.
Despite this shortening, a half-foot of chromatin is still much too long to fit into the nucleus, which is typically only
10 to 20 microns in diameter. Therefore, chromatin is further coiled into an even shorter, thicker fiber, termed the
"30-nanometer fiber," because it is approximately 30 nanometers in diameter.
Over the years, there has been a great deal of speculation concerning the manner in which nucleosomes are folded
into 30-nanometer fibers. Part of the problem lies in the fact that electron microscopy is perhaps the best way to
visualize packaging, but individual nucleosomes are hard to discern after the fiber has formed. In addition, it also
makes a difference whether observations are made using isolated chromatin fibers or chromatin within whole nuclei.
Thus, the 30-nanometer fiber may be highly irregular and not quite the uniform structure. Interestingly, histone H1
is very important in stabilizing chromatin higher-order structures, and 30-nanometer fibers form most readily when
H1 is present.

Processes such as transcription and replication require the two strands of DNA to come apart temporarily, thus
allowing polymerases access to the DNA template. However, the presence of nucleosomes and the folding of
chromatin into 30-nanometer fibers pose barriers to the enzymes that unwind and copy DNA. It is therefore
important for cells to have means of opening up chromatin fibers and/or removing histones transiently to permit
transcription and replication to proceed. Generally speaking, there are two major mechanisms by which chromatin is
made more accessible:

• Histones can be enzymatically modified by the addition of acetyl, methyl, or phosphate groups.
• Histones can be displaced by chromatin remodeling complexes, thereby exposing underlying DNA
sequences to polymerases and other enzymes.

It is important to remember that these processes are reversible, so modified or remodeled chromatin can be returned
to its compact state after transcription and/or replication are complete.

Electron micrograph of chromatin

ORGANISATION OF PROKARYOTIC GENOME:
• Genetic material in a cell: All cells have the capability to give rise to new cells and the encoded information in a
living cell is passed from one generation to another. The information encoding material is the genetic or hereditary
material of the cell.

• Prokaryotic genetic material: The prokaryotic (bacterial) genetic material is usually concentrated in a specific clear
region of the cytoplasm called nucleiod.

• The bacterial chromosome is a single, circular, double stranded DNA molecule mostly attached to the plasma
membrane at one point. It does not contain any histone protein.

• Escherichia coli DNA is circular molecule 4.6 million base pairs in length, containing 4288 annotated protein-
coding genes (organized into 2584 operons), seven ribosomal RNA (rRNA) operons, and 86 transfer RNA (tRNA)
genes.

• Certain bacteria like the Borrelia burgdorferi possess array of linear chromosome like eukaryotes.

• Besides the chromosomal DNA many bacteria may also carry extra chromosomal genetic elements in the form of
small, circular and closed DNA molecules, called plasmids.

• They generally remain floated in the cytoplasm and bear different genes based on which they have been
studied.

• Some of the different types of plasmids are F plasmids, R plasmids, virulent plasmids, metabolic plasmids
etc.

E. coli: A Model Prokaryote

• Much of what is known about prokaryotic chromosome structure was derived from studies of Escherichia coli, a

bacterium that lives in the human colon and is commonly used in laboratory cloning experiments.

• In the 1950s and 1960s, this bacterium became the model organism of choice for prokaryotic research when a
group of scientists used phase-contrast microscopy and autoradiography to show that the essential genes of E.
coli are encoded on a single circular chromosome packaged within the cell nucleoid.
• Prokaryotic cells do not contain nuclei or other membrane-bound organelles. In fact, the word "prokaryote"
literally means "before the nucleus." The nucleoid is simply the area of a prokaryotic cell in which the chromosomal
DNA is located.

• This arrangement is not as simple as it sounds, however, especially considering that the E.coli chromosome is
several orders of magnitude larger than the cell itself. So, if bacteria chromosomes are so huge, how can they fit
comfortably inside a cell—much less in one small corner of the cell?

DNA Supercoiling

• The answer to the question asked lies in DNA packaging.

• Whereas eukaryotes wrap their DNA around proteins called histones to help package the DNA into smaller spaces,
most prokaryotes do not have histones (with the exception of those species in the domain Archaea).

• Thus, one way prokaryotes compress their DNA into smaller spaces is through supercoiling.

• Imagine twisting a rubber band so that it forms tiny coils. Now twist it even further, so that the original coils fold
over one another and form a condensed ball. When this type of twisting happens to a bacterial genome, it is known
as supercoiling.

• Genomes can be negatively supercoiled, meaning that the DNA is twisted in the opposite direction of the double
helix, or positively supercoiled, meaning that the DNA is twisted in the same direction as the double helix.

• Most bacterial genomes are negatively supercoiled during normal growth.

Prokaryotic genome organization through:

1. Nucleoid (protein DNA interaction)

2. Supercoiling
3. DNA loops
Prokaryotic DNA must be tightly packed so that it can fit in the nucleoid. DNA packing is achieved through coiling,
compacting and supercoiling.

(A) The circular chromosomal DNA molecule can be compacted through (B) the formation of looped
structures.
(C) The looped DNA can be further compacted by DNA supercoiling. Note: the coloured balls represent
proteins involved in supercoilng.

Proteins Involved in Supercoiling

• During the 1980s and 1990s, researchers discovered that multiple proteins act together to fold and condense
prokaryotic DNA.

• In particular, one protein called HU, which is the most abundant protein in the nucleoid, works with an enzyme
called topoisomerase I to bind DNA and introduce sharp bends in the chromosome, generating the tension necessary
for negative supercoiling.

• Recent studies have also shown that other proteins, including integration host factor (IHF), can bind to specific
sequences within the genome and introduce additional bends.

• The folded DNA is then organized into a variety of conformations that are supercoiled and wound around
tetramers of the HU protein, much like eukaryotic chromosomes are wrapped around histones.

• Once the prokaryotic genome has been condensed, DNA topoisomerase I, DNA gyrase, and other proteins help
maintain the supercoils. One of these maintenance proteins, H-NS, plays an active role in transcription by
modulating the expression of the genes involved in the response to environmental stimuli.

• Another maintenance protein, factor for inversion stimulation (FIS), is abundant during exponential growth and
regulates the expression of more than 231 genes, including DNA topoisomerase I.
Accessing supercoiled genes:
It has been determined that prokaryotic DNA replication occurs at a rate of 1,000 nucleotides per second, and
prokaryotic transcription occurs at a rate of about 40 nucleotides per second. Researchers have noted that the
nucleoid usually appears as an irregularly shaped mass within the prokaryotic cell, but it becomes spherical when
the cell is treated with chemicals to inhibit transcription or translation. Moreover, during transcription, small regions
of the chromosome can be seen to project from the nucleoid into the cytoplasm (i.e., the interior of the cell), where
they unwind and associate with ribosomes, thus allowing easy access by various transcriptional proteins. These
projections are thought to explain the mysterious shape of nucleoids during active growth. When transcription is
inhibited, however, the projections retreat into the nucleoid, forming the aforementioned spherical shape.

Because there is no nuclear membrane to separate prokaryotic DNA from the ribosomes within the cytoplasm,
transcription and translation occur simultaneously in these organisms. This is strikingly different from eukaryotic
chromosomes, which are confined to the membrane-bound nucleus during most of the cell cycle. In eukaryotes,
transcription must be completed in the nucleus before the newly synthesized mRNA molecules can be transported to
the cytoplasm to undergo translation into proteins.

Difference between Prokaryotic and Eukaryotic chromosomes:

GENOME ORGANISATION IN VIRUSES:
Viruses have either DNA or RNA genomes that can be either single-stranded or double stranded. Thus, compared
with cells, viral genomes can create some unusual challenges for genetic information flow.

The Baltimore Scheme: DNA Viruses – Given by David Baltimore

The scheme is based on the relationship of the viral genome to its mRNA and recognizes seven classes of viruses;
three classes have DNA genomes and four have RNA genomes. By convention in virology, viral mRNA is always
considered to be of the plus (+) configuration. Thus, to understand the molecular biology of a particular class of
virus, one must know the nature of the viral genome and the steps necessary to produce (+) mRNA from it.

CLASSES OF DNA VIRUSES: There are three classes of DNA viruses under Baltimore classification.

Baltimore class I: dsDNA (+/-) virus

• Double-stranded DNA viruses are in Baltimore class I.

• The mechanism of mRNA production and genome replication of class I viruses is the same as that used by cells.

• The (-) strand of the ds DNA will serve as the template during transcription for the formation of (+) mRNA.

• Examples of Class I viruses include Herpesviridae, Adenoviridae, Papoviridae and bacteriophage T4.
 mRNA production and genome replication of Class I

Baltimore class II: (+) ssDNA virus

• A virus containing a single-stranded genome may be either a positive-strand virus (also called a plus-strand virus)
or a negative-strand virus (also called a minus-strand virus).

• Class II viruses contain single-stranded plus strand DNA genomes. Transcription of such a genome would yield (-)
mRNA, and thus, before (+) mRNA can be produced from class II viruses, a complementary DNA strand must first
be made to form a double-stranded DNA intermediate; this is called the replicative form.

• The replicative form can be used for rolling-circle replication, where one strand is nicked and replication
enzymes are used to extend the free 3’ end. As a complementary strand is synthesized around the circular DNA, the
5’ end is peeled off, leading to a displaced strand that continues to grow in length.
 Rolling circle replication in phage φ X174

• The latter is used to produce (+) mRNA, and as the source of new genome copies.

• The plus strand becomes the genome while the minus strand is discarded.

• With only one known exception, all single-stranded DNA viruses are positive-strand viruses.

• Examples of Class II viruses are Anelloviridae, Circoviridae, Parvoviridae and φ X174

mRNA production and genome replication of Class II

Baltimore class VII: dsDNA (+/-) virus
• The DNA genome is partially double-stranded, but contains a single-stranded region.

• After gaining entrance into the cell’s nucleus, host cell enzymes are used to fill in the gap with complementary
bases to form a dsDNA closed loop.

• Gene transcription yields a plus-strand RNA known as the pregenome, as well as the viral enzyme reverse
transcriptase, an RNA-dependent DNA-polymerase.

• The pregenome is used as a template for the reverse transcriptase to produce minus-strand DNA genomes, with a
small piece of pregenome used as a primer to produce the double-stranded region of the genomes.

CLASSES OF RNA:
There are four classes of RNA viruses under Baltimore classification. The production of mRNA and genome
replication will obviously be different for RNA viruses than for DNA viruses. Cellular RNA polymerases do not
catalyze the formation of RNA from an RNA template, but instead require a DNA template. Therefore, depending
on the virus, RNA viruses must either carry in their virions or encode in their genomes an RNA-dependent RNA
polymerase called RNA replicase. RNA replicases replicate the viral RNA genome and produce viral-specific
mRNA.

Baltimore class III: dsRNA (+/-) virus

• RNA replicase must synthesize a plus strand of RNA from the negative-strand template, and the plus strand is then
used as mRNA.

• RNA replicase also synthesizes the (-) strand of the RNA, which with the (+) strand makes up the RNA genome.

mRNA production and genome replication of Class III

Baltimore class IV: ssRNA (+) virus
• Class IV ssRNA viruses have positive-sense RNA genomes, meaning they can be directly read by ribosomes to
translate into proteins.

• They are further divided into viruses with polycistronic mRNA and those with complex transcription.

• Polycistronic mRNA is translated into a polyprotein that is subsequently cleaved to form separate proteins.

• Viruses with complex transcription use ribosomal frameshifting and proteolytic processing to produce multiple
proteins from the same gene sequences.

• Examples of some Class IV viruses are Coronaviridae, Flaviviridae, Astroviridae, and Picornaviridae.

Translation and genome replication in Class IV

Baltimore class V: ssRNA (-) virus
• For negative-strand RNA viruses, RNA replicase must synthesize a plus strand of RNA from the negative-strand
template, and the plus strand is then used as mRNA.

• The plus strand is also used as a template to make more negative-strand genomes.

mRNA production and genome replication of class V

Baltimore class VI: ssRNA (+), retroviruses
• Retroviruses are animal viruses whose genomes consist of single stranded RNA of the plus configuration but
replicate through a double-stranded DNA intermediate.

• The process of copying the information found in RNA into DNA is called reverse transcription and is catalyzed
by an enzyme called reverse transcriptase.

• Human immunodeficiency virus (HIV, the causative agent of AIDS) is a retrovirus.

mRNA production and genome replication of class VI

BIBLIOGRAPHY:

Ncbi.nlm.nih.gov

Ncbi.nlm.nih/pubmed

Nature.com

Bio.libretexts.org

Prescott’s Microbiology – 11th Edition