Lect-04

Prokaryotic and
eukaryotic chromosome
structure
 Learning Outcomes (LO)
After students learn this lecture, students are able to
explain:
1. Prokaryotic chromosome structure (LO 4.1)
2. Chromatin structure (LO 4.2)
3. Eukaryotic chromosome structure (LO 4.3)
4. Genome complexity (LO 4.4)
5. The flow of genetic information (LO 4.5)
 PROKARYOTIC CHROMOSOME STRUCTURE

The Escherichia coli chromosome

 Prokaryotic genomes are exemplified by the E. coli chromosome.
 The bulk of the DNA in E. coli cells consists of a single closed-circular
DNA molecule of length 4.6 million base pairs.
 The DNA is packaged into a region of the cell known as the nucleoid.
 This region has a very high DNA concentration, perhaps 30–50 mg ml–1,
as well as containing all the proteins associated with DNA, such as
polymerases, repressors and others.
 A fairly high DNA concentration in the test tube would be 1 mg ml–1.
 In normal growth, the DNA is being replicated continuously and there
may be on average around two copies of the genome per cell, when
growth is at the maximal rate.

LO 4.1: Students are able to explain Prokaryotic chromosome structure

  Experiments in which DNA from E. coli is carefully isolated free of most of the attached
proteins and observed under the electron microscope reveal one level of organization of
the nucleoid.
 The DNA consists of 50–100 domains or loops, the ends of which are constrained by
binding to a structure which probably consists of proteins attached to part of the cell
membrane (Fig. 1).
 The loops are about 50–100 kb in size.
 It is not known whether the loops are static or dynamic, but one model suggests that the
DNA may spool through sites of polymerase or other enzymic action at the base of the
loops.

LO 4.1: Students are able to explain Prokaryotic chromosome structure

 PROKARYOTIC CHROMOSOME STRUCTURE
Supercoiling of the genome
 The E. coli chromosome as a whole is negatively
supercoiled, although there is some evidence that individual
domains may be supercoiled independently(Fig. 1).

LO 4.1: Students are able to explain Prokaryotic chromosome structure

 PROKARYOTIC CHROMOSOME STRUCTURE
DNA-binding proteins
 The looped DNA domains of the chromosome are constrained
further by interaction with a number of DNA-binding proteins.
 The most abundant of these are protein HU, a small basic
(positively charged) dimeric protein, which binds DNA
nonspecifically by the wrapping of the DNA around the protein,
and H-NS (formerly known as protein H1), a monomeric
neutral protein, which also binds DNA nonspecifically in terms
of sequence, but which seems to have a preference for
regions of DNA which are intrinsically bent.
 These proteins are sometimes known as histone-like proteins,
and have the effect of compacting the DNA, which is essential
for the packaging of the DNA into the nucleoid, and of
stabilizing and constraining the supercoiling of the
chromosome.
LO 4.1: Students are able to explain Prokaryotic chromosome structure
 CHROMATIN STRUCTURE
Chromatin
 The total length of DNA in a eukaryotic cell depends on the species, but it can be
thousands of times as much as in a prokaryotic genome, and is made up of a
number of discrete bodies called chromosomes (46 in humans).
 The DNA in each chromosome is believed to be a single linear molecule, which
can be up to several centimeters long.
 All this DNA must be packaged into the nucleus, a space of approximately the
same volume as a bacterial cell; in fact, in their most highly condensed forms, the
chromosomes have an enormously high DNA concentration of perhaps 200 mg
ml–1.
 This feat of packing is accomplished by the formation of a highly organized
complex of DNA and protein, known as chromatin, a nucleoprotein complex.
 More than 50% of the mass of chromatin is protein.
 Chromosomes greatly alter their level of compactness as cells progress through
the cell cycle, varying between highly condensed chromosomes at metaphase
(just before cell division), and very much more diffuse structures in interphase.
 This implies the existence of different levels of organization of chromatin.

CP 4.2: Students are able to explain chromatin structure

 CHROMATIN STRUCTURE
Histones
 Most of the protein in eukaryotic chromatin consists of
histones, of which there are five families, or classes: H2A,
H2B, H3 and H4, known as the core histones, and H1.
 The core histones are small proteins, with masses between
10 and 20 kDa, and H1 histones are a little larger at around
23 kDa.
 All histone proteins have a large positive charge; between 20
and 30% of their sequences consist of the basic amino
acids, lysine and arginine.
 This means that histones will bind very strongly to the
negatively charged DNA in forming chromatin.

CP 4.2: Students are able to explain chromatin structure

 CHROMATIN STRUCTURE
Nucleosomes & The role of H1
 The nucleosome core is the basic unit
of chromosome structure, consisting of
a protein octamer containing two each
of the core histones, with 146 bp of
DNA wrapped 1.8 times in a left-
handed fashion around it.
 A single molecule of H1 stabilizes the
DNA at the point at which it enters and
leaves the nucleosome core, and
organizes the DNA between
nucleosomes.
 A nucleosome core plus H1 is known
as a chromatosome.
 In some cases, H1 is replaced by a
variant, H5, which binds more tightly,
and is associated with DNA which is
inactive in transcription.

CP 4.2: Students are able to explain chromatin structure

 CHROMATIN STRUCTURE
Linker DNA
 The linker DNA between
the nucleosome cores
varies between less than 10
and more than 100 bp, but
is normally around 55 bp.
 The nucleosomal repeat
unit is hence around 200
bp.

CP 4.2: Students are able to explain chromatin structure

 CHROMATIN STRUCTURE
The 30 nm fiber
 Chromatin is organized into a
larger structure, known as the
30 nm fiber or solenoid,
thought to consist of a left-
handed helix of nucleosomes
with approximately six
nucleosomes per helical turn.
 Most chromatin exists in this
form.

CP 4.2: Students are able to explain chromatin structure

 CHROMATIN STRUCTURE
Higher order structure
 On the largest scale, chromosomal DNA is organized into
loops of up to 100 kb in the form of the 30 nm fiber,
constrained by a protein scaffold, the nuclear matrix.
 The overall structure somewhat resembles that of the
organizational domains of prokaryotic DNA.

CP 4.2: Students are able to explain chromatin structure

 EUKARYOTIC CHROMOSOME STRUCTURE
The mitotic chromosome
 The classic picture of paired sister
chromatids at mitosis represents the
most highly condensed state of
chromatin.
 The linear DNA traces a single path
from one tip of the chromosome to
the other, in successive loops of up
to 100 kb of 30 nm fiber anchored to
the nuclear matrix in the core.

LO 4.3: Students are able to explain eukaryotic chromosome structure

 EUKARYOTIC CHROMOSOME STRUCTURE
The centromere
 The centromere is the region
where the two chromatids are
joined and is also the site of
attachment, via the
kinetochore, to the mitotic
spindle, which pulls apart the
sister chromatids at anaphase.
 Centromeres are
characterized by specific short
DNA sequences although, in
mammalian cells, there may
be an involvement of satellite
DNA.

LO 4.3: Students are able to explain eukaryotic chromosome structure

 EUKARYOTIC CHROMOSOME STRUCTURE
Telomeres
 Telomeres are specialized DNA sequences that form the
ends of the linear DNA molecules of the eukaryotic
chromosomes.
 A telomere consists of up to hundreds of copies of a short
repeated sequence (5-TTAGGG-3 in humans), which is
synthesized by the enzyme telomerase (an example of a
ribonucleoprotein) in a mechanism independent of normal
DNA replication.
 The telomeric DNA forms a special secondary structure, the
function of which is to protect the ends of the chromosome
proper from degradation.
 Independent synthesis of the telomere acts to counteract the
gradual shortening of the chromosome resulting from the
inability of normal replication to copy the very end of a linear
DNA molecule.
LO 4.3: Students are able to explain eukaryotic chromosome structure
 EUKARYOTIC CHROMOSOME STRUCTURE
Interphase chromosomes
 In interphase, the genes on the chromosomes are being
transcribed and DNA replication takes place (during S-
phase).
 During this time, which is most of the cell cycle, the
chromosomes adopt a much more diffuse structure and
cannot be visualized individually.
 It is believed, however, that the chromosomal loops are still
present, attached to the nuclear matrix.

LO 4.3: Students are able to explain eukaryotic chromosome structure

 EUKARYOTIC CHROMOSOME STRUCTURE
Heterochromatin
 Heterochromatin comprises a portion of the chromatin in
interphase which remains highly compacted, although not so
compacted as at metaphase.
 It can be visualized under the microscope as dense regions
at the periphery of the nucleus, and probably consists of
closely packed regions of 30 nm fiber.
 It has been shown more recently that heterochromatin is
transcriptionally inactive.
 It is believed that much of the heterochromatin may consist
of the repeated satellite DNA close to the centromeres of the
chromosomes, although in some cases entire chromosomes
can remain as heterochromatin, for example one of the two
X chromosomes in female mammals.

LO 4.3: Students are able to explain eukaryotic chromosome structure

 EUKARYOTIC CHROMOSOME STRUCTURE
Euchromatin
 The rest of the chromatin, which is not
visible as heterochromatin, is known
historically by the catch-all name of
euchromatin, and is the region where
all transcription takes place.
 Euchromatin is not homogeneous,
however, and is comprised of relatively
inactive regions, consisting of
chromosomal loops compacted in 30
nm fibers, and regions (perhaps 10%
of the whole) where genes are actively
being transcribed or are destined to be
transcribed in that cell type, where the
30 nm fiber has been dissociated to
the ‘beads on a string’ structure.

 Parts of these regions may be depleted of nucleosomes altogether, particularly

within promoters, to allow the binding of transcription factors and other proteins
(Fig. 2).
LO 4.3: Students are able to explain eukaryotic chromosome structure
 EUKARYOTIC CHROMOSOME STRUCTURE
DNase I hypersensitivity
 Active regions of
chromatin, or regions
where the 30 nm fiber is
interrupted by the
binding of a specific
protein to the DNA, or
by ongoing transcription,
are characterized by
hypersensitivity to
deoxyribonuclease I
(DNase I).

LO 4.3: Students are able to explain eukaryotic chromosome structure

 EUKARYOTIC CHROMOSOME STRUCTURE
CpG methylation
 5’-CG-3’ (CpG) sequences in mammalian DNA are normally
methylated on the cytosine base; however, ‘islands’ of
unmethylated CpG occur near the promoters of frequently
transcribed genes, and form regions of particularly high
DNase I sensitivity.

LO 4.3: Students are able to explain eukaryotic chromosome structure

 EUKARYOTIC CHROMOSOME STRUCTURE
Histone variants and modification
 The major mechanisms for the condensing and decondensing of
chromatin are believed to operate directly through the histone proteins
which carry out the packaging.
 Short-term changes in chromosome packing during the cell cycle seem to
be modulated by chemical modification of the histone proteins.
 For example, actively transcribed chromatin is associated with the
acetylation of lysine residues in the N-terminal regions of the core
histones, whereas the condensation of chromosomes at mitosis is
accompanied by the phosphorylation of histone H1.
 These changes alter the positive charge on the histone proteins, and may
affect the stability of the various chromatin conformations, for example the
30 nm fibers or the interactions between them.
 Longer term differences in chromatin condensation are associated with
changes due to stages in development and different tissue types.
 These changes are associated with the utilization of alternative histone
variants, which may also act by altering the stability of chromatin
conformations.

LO 4.3: Students are able to explain eukaryotic chromosome structure

 GENOME COMPLEXITY
Noncoding DNA
 This technique allows different classes of repeated DNA to
be identified by the effect of multiple copies of a sequence
on the rate of renaturation of denatured genomic DNA
fragments.
 By this method, human DNA can be classified into highly
repetitive DNA, moderately repetitive DNA and unique DNA.

LO 4.4: Students are able to explain genome complexity

 GENOME COMPLEXITY
Reassociation kinetics
 This technique allows
different classes of
repeated DNA to be
identified by the effect of
multiple copies of a
sequence on the rate of
renaturation of denatured
genomic DNA fragments.
 By this method, human
DNA can be classified
into highly repetitive DNA,
moderately repetitive
DNA and unique DNA.

LO 4.4: Students are able to explain genome complexity

 GENOME COMPLEXITY
Unique sequence DNA
 This fraction of genomic DNA is the slowest to reassociate
on a C0t curve, and corresponds to the coding regions of
genes which occur in only one or a few copies per haploid
genome, and any unique intervening sequence.
 In the E. coli genome, virtually all the DNA has a unique
sequence, since it consists predominantly of more or less
contiguous (adjacent) single-copy genes.
 However, since E. coli has approximately 1000 times less
DNA than a human cell, any given sequence has
correspondingly more alternative partners at a given
concentration, and its reassociation occurs faster, that is at a
lower value of C0t.

LO 4.4: Students are able to explain genome complexity

 GENOME COMPLEXITY
Tandem gene clusters
 Moderately repetitive DNA consists of a number of types of repeated sequence.
 At the lower end of the repeat scale come genes which occur as clusters of
multiple repeats.
 These are genes whose products are required in unusually large quantities.
 One example is the rRNA-encoding genes (rDNA).
 The gene which encodes the 45S precursor of the 18S, 5.8S and 28S rRNAs, for
example, is repeated in arrays containing from around 10 to around 10 000
copies depending on the species.
 In humans, the 45S gene occurs in arrays
on five separate chromosomes, each
containing around 40 copies.
 In interphase, these regions are spatially
located together in the nucleolus, a dense
region of the nucleus, which is a factory
for rRNA production and modification.

 A second example of tandem gene clusters is given by the histone genes, whose
products are produced in large quantities during S-phase.
 The five histone genes occur together in a cluster, which is directly repeated up to
several hundred times in some species.
LO 4.4: Students are able to explain genome complexity
 GENOME COMPLEXITY
Dispersed repetitive DNA
 Much of the moderately repetitive DNA comprises a number
of DNA sequences of a few hundred base pairs (SINES, or
short interspersed elements) or between one and five
thousand base pairs (LINES, or long interspersed elements),
each repeated more than 100 000 times and scattered
throughout the genome.
 The most prominent examples in humans are the Alu and the
L1 elements, which may be parasitic DNA sequences,
replicating themselves by transposition.

LO 4.4: Students are able to explain genome complexity

 GENOME COMPLEXITY
Satellite DNA
 Satellite DNA, which occurs mostly near the centromeres of
chromosomes, and may be involved in attachment of the
mitotic spindle, consists of huge numbers of tandem repeats
of short (up to 30 bp) sequences.
 Hypervariability in satellite DNA is the basis of the DNA
fingerprinting technique.
 Figure 2 shows an example of a Drosophila satellite DNA
sequence, which occurs millions of times in the insect’s
genome.

LO 4.4: Students are able to explain genome complexity

 GENOME COMPLEXITY
Genetic polymorphism
 Base changes (mutations) in a gene or a chromosomal locus
can create multiple forms (polymorphs) of that locus which is
then said to show genetic polymorphism.
 The term can describe different alleles of a single copy gene
in a single individual as well as the different sequences
present in different individuals in a population.
 Common types are single nucleotide polymorphisms (SNPs)
and simple sequence length polymorphisms (SSLPs).
 Where SNPs create or destroy the sequence recognized by a
restriction enzyme, restriction fragment length polymorphism
(RFLP) will result.

LO 4.4: Students are able to explain genome complexity

 THE FLOW OF GENETIC INFORMATION
The central dogma
 The central dogma is the original
proposal that ‘DNA makes RNA
makes protein’, which happen via
the processes of transcription and
translation respectively.
 This is broadly correct, although a
number of examples are known
which contradict parts of it.
 Retroviruses reverse transcribe RNA
into DNA, a number of viruses are
able to replicate RNA directly into an
RNA copy, and a number of
organisms can edit a messenger
RNA sequence so that the protein
coding sequence is not directly
specified by DNA sequence.

LO 4.5: Students are able to explain the flow of genetic information

 THE FLOW OF GENETIC INFORMATION
Prokaryotic gene expression
 Transcription of a single gene or an operon starts at the
promoter, ends at the terminator and produces a
monocistronic or polycistronic messenger RNA.
 The coding regions of the message are translated by the
ribosome from the start codon (close to the ribosome binding
site) to the stop codon.
 Transfer RNAs deliver the appropriate amino acid, according
to the genetic code, to the growing protein chain.

LO 4.5: Students are able to explain the flow of genetic information

 THE FLOW OF GENETIC INFORMATION
Eukaryotic gene expression
 In most cases monocistronic messenger RNAs are
transcribed from a gene, initiated at a promoter.
 The resulting pre-messenger RNA is capped at the 5′-end
and has a poly(A) tail added to the 3′-end.
 Introns are removed by splicing before the mature mRNA is
exported from the nucleus to be translated by ribosomes in
the cytoplasm.

LO 4.5: Students are able to explain the flow of genetic information

DNA replication