Organisation of Genomes - Eukaryotes 

Non-dividing cells  Dividing cells 

Each chromosome comprise one DNA molecule which exists as  After S phase, each chromosome comprise two DNA molecules which 
decondensed chromatin   exists as 
● Euchromatin is decondensed and transcriptionally active   ● Decondensed chromatin after S phase and before prophase 
● Heterochromatin is not fully decondensed and is  ● 2 identical sister chromatids (condensed form) during nuclear 
transcriptionally inactive   division  

Packing of DNA in chromosomes 

First level of condensation   DNA is packed into nucleosome to produce the 10nm chromatin fibre (nucleosome fibre) 
 
A nucleosome consists of DNA wound 1 ¾ times around a protein core composed of an octamer of 4 
histone molecules: H2A, H2B, H3, H4 (two of each) 
● Histones have a high concentration of basic residues (positively charged) which interact with 
negatively charged sugar-phosphate backbone of DNA 
 
Beads-on-string look 
● Each bead is a nucleosome  
● String is called linker DNA 
 
Histones leave the DNA briefly during DNA replication and gene expression processes that require access 
to DNA 
Second level of  DNA is further coiled and folded by histone H1 and linker DNA to produce 30nm chromatin fibre 
condensation   (solenoid) 

Euchromatin 
DNA associated l​ oosely​ with histone proteins → adopts confirmation of nucleosome fibre 
 
Heterochromatin 
DNA associated t​ ightly ​with histone proteins → adopts confirmation of solenoid 

Third level of condensation   Non-histone chromosomal proteins form a scaffold involved in condensing 30nm chromatin fibre into 
loops called looped domains, forming 300 nm chromatin fibre  
 
Further fold and coil to produce metaphase chromosome 
● Width of one chromatid: 700nm 
 
Particular genes always end up located at same places → packing is highly specific and precise 

Role of condensation  
1. Organise and pack giant DNA molecule into more compact structure 
2. Significance of packing into chromatin 
a. To fit within the cell’s nucleus 
b. To prevent long DNA molecules from getting tangled 
3. Significance of packing into chromosomes during cell division  
a. To facilitate segregation into daughter nuclei 
b. WIll not be entangled and break during separation at anaphase 
i. If break, will lead to mutations and non-disjunction  
 Organization of DNA in chromosome 

Coding sequences  Non-coding sequences  

Carry information corresponding  Codes for 
to specific genes   ● Introns 
  ● 5’ and 3’ untranslated regions 
Heavily used gene products (Eg.  ● Promoters 
histones, rRNA) are encoded by  ● Enhancer and silencers sequences 
multiple copies of genes  ● Replication origin and termination  
arranged in repeats, either  ● Centromeres and telomeres 
● Interspersed (separated   
by other sequences)  Repeated sequences 
● In tandem (appear one  ● Tandemly repetitive DNA 
after another)  ○ Satellite DNA​ have a core sequence of 5 to 171 bp with total lengths between 100kb to 
several Mb 
○ Minisatellites​ have core sequence of 20 bp with total lengths between 100 and 20000 
bp 
○ Microsatellites​ have core sequence of 2 to 4 bp tandemly repeated between 10 to 20 
times 
○ Examples: centromeres and telomeres 
● Interspersed repetitive DNA  
○ Examples: promoters, enhancers and origins of replication  

   
Centromere 
Structure   Function  

AT rich region   Binds to kinetochore proteins which has DNA binding site complementary to 
shape of centromere 
 
Spindle fibres bind to kinetochore  
 

Telomere   
Structure  Function 

Short, repeated  1. Prevent fusion of ends of chromosomes with other DNA molecules 
TG rich  2. Prevent exonucleases from degrading ends of linear DNA molecules 
sequences  3. Prevent loss of genetic information by acting as a disposable buffer 
4. Facilitate replication of ends of DNA molecules without loss of termini 
5. Prevent cell cycle arrest as specific proteins associated with telomeric 
DNA prevent staggered ends from activating the cell system for 
monitoring DNA damage 
 
Role of telomerase  
Lengthen telomere 
 Comparing structure/organisation of prokaryotic and eukaryotic chromosomes 

Eukaryotic chromosomes  Prokaryotic chromosomes 

Linear Circular

Tens to hundreds of millions of base pairs in length Few million base pairs in length

Occur in sets (Eg. diploid organisms have 2 sets of Single type of chromosome
chromosomes)
Several thousand different genes interspersed
Several hundred and thousand different genes interspersed
One origin of replication
Many origin of replication
Control of Gene Expression - Eukaryotes  

Availability of Genes for Transcription 

Histone modification   Long-term changes occur during development as chromatin goes from diffused to condensed or vice versa 
 
Transcription initiation is prevented if the promoter is part of a nucleosome. Thus, activation of genes for 
transcription require changes in the state of chromatin, called chromatin remodelling. 
 
Histone tails protruding outward from the nucleosome are accessible to various modifying enzymes which 
catalyse the addition or removal of specific chemical groups  
 
Histone acetylation ​Up-regulates gene expression 
● Acetyl groups are attached to lysines in histone tails, catalysed by histone acetyltransferase (HAT) 
● Positive charges of lysine is neutralised and histone tails cannot interact with neighbouring nucleosomes 
○ Electrostatic interaction between neighbouring nucleosomes promotes folding of chromosome 
into more compact structure 
● Chromatin has more diffused structure → transcription factors have easier access to genes 
● HAT also binds to and aids in recruitment of transcription machinery 
 
Histone Methylation D ​ own-regulates gene expression 
● Addition of methyl groups promote condensation of chromatin 
● Promotes binding of other proteins to methyl groups → structure become unavailable for other 
proteins/enzymes to bind  

   
DNA Methylation  Cytosine residues in GC-rich sequences near or in the promoter region are methylated to produce 
Down-regulates gene  5-methylcytosine  
expression   
More heavily methylated genes are not expressed 
 
Proteins that bind to methylated DNA recruit deacetylation enzymes  
 
Responsible for long term inactivation of genes 
 
At DNA sites where one strand is methylated, methylation enzymes correctly methylate daughter DNA molecule 
after each round of DNA replication  
● Methylation patterns are passed on  

Transcriptional Control 

Control elements   Promoter 

Core promoter  Proximal control elements  

Contains transcription start site and TATA box (upstream of former)  Located immediately upstream of core 
  promoter 
Determines basal level of transcription    
  Contain CAAT box and GC-rich 
TATA box is an AT-rich sequence which serves as binding site of  sequences which determine frequency 
general transcription factors that facilitate binding of RNA polymerase II  of transcription events occurring 
to transcription start site    
  Control frequency of T1 
TATA box binding protein (TBP) has a DNA binding site 
 complementary in shape to TATA box. Upon binding, DNA is distorted 
(Refer to image)  

 
● Promotes recruitment of other transcription factors  
● Reveal more of promoter → RNA polymerase can bind to more 
easily  
 
Distal control elements​ C
​ ontrol rate of T1 
Enhancers   Silencers 

Can exert their effects even when located hundred or thousands of bases away from transcription units 
 
Found in a variety of locations; upstream and downstream of transcription units or within transcribed portions 
  
Function in an orientation-independent fashion  

Binding site for specific transcription factors called activators  Binding site for specific transcription factors 
● Increases rate of transcription   called repressors 
  ● Inhibits or decreases rate of 
A gene can have multiple enhancers, each active at different time  transcription  
or in different cell type of locations 
 

Transcription  General transcription factors  

factors  Protein-protein interactions crucial for initiation. Only when the complete initiation complex has assembled can the 
RNA polymerase move along template strand, producing complementary strand of RNA  
 
Interaction of general transcription factors and RNA polymerase leads to low rate of initiation and production of few 
RNA transcripts  
 
Specific transcription factors 
Activators  Repressors 

Increases rate by  Decrease rate by 

● Recruits DNA bending protein 
○ DNA is bent, bringing enhancer 
sequence nearer to mediator 
proteins 
● Recruits a group of mediator proteins 
○ Through protein-protein 
interactions, help RNA polymerase 
and transcription factors assemble 
 at promoter 
● Facilitate correct positioning of transcription 
initiation complex on promoter for 
transcription to occur  
 
Two common structures 
● A DNA binding domain 
● One or more activation domains which 
binds other regulatory proteins, facilitating 
protein-protein interactions that result in 
transcription  

 
(A) Only one can bind at one time due to steric hindrance  
(B) Activation surface is required for activator to recruit 
mediator proteins and interact with other proteins to 
assemble GTF  
(C) Binds to GTF, preventing other GTF from binding to it → 
cannot form transcription initiation complex 
 
 Post-transcriptional control 

Alternation of mRNA ends  5’ end is capped off with a modified form of guanine nucleotide 
● Protect mRNA from degradation by hydrolytic enzymes 
● An attach here sign for ribosomes 
 
At 3’ end, enzyme makes a poly(A) tail consisting of 50-250 adenine nucleotides 
● Inhibits degradation and helps ribosomes attach to mRNA 
● Facilitate export of mRNA from nucleus  

RNA Splicing  Introns are cut out and exons joined together to form an mRNA molecule with continuous coding 
sequence  
 
Mechanism  
● Signals for RNA splicing are short nucleotide sequences at ends of introns known as 
splice sites 
● Small nuclear ribonucleoproteins (snRNPs) recognise the splice sites 
● Several snRNPs join with additional proteins to form a spliceosome 
○ Cuts at specific points to release introns and immediately joins exons 
 
Does mutation in intron affect the resulting sequence during T2? 
- Yes, mutation can remove or introduce splice sites 
- Resultant mRNA is longer or shorter than what it is supposed to be → non-functional 
protein 
 
Significance  
Alternative splicing  
● pre-mRNA can be spliced in more than one way  
● A single gene can give rise to 2 or more polypeptide, with variation in their functions 
○ Helps to make our genome compact (don’t need one gene sequence for every 
protein) 
 Translational Control 

mRNA stability  Half-life of mRNA  

● Longer half-life → stay in cytosol longer → used for times as template → more protein synthesised  
 
Factors affecting stability: 
Length of poly(A) tail  Destabilizing sequences   Binding of regulatory proteins to 
3’ UTR 

Binding of poly(A)-binding protein  Most commonly found at 3’ UTR  Stabilize mRNA by preventing 
enhances RNA stability    degradation by endonucleases  
  Eg. AU-rich element (ARE)   
As mRNA ages, poly(A) tail is shortened  ● Consensus sequence  Eg. Iron regulatory protein (IRP) 
by cellular exonucleases  AUUUA is recognised by  binds at iron response element 
  cellular proteins that bind  (IRE) 
Once the poly(A) tail becomes too short,  to ARE, influencing whether   
poly(A)-binding protein can no longer  or not mRNA is rapidly   
bind   degraded 
 
Enzymatic shortening triggers actions of 
enzymes which remove the 5’ cap 
 
Nucleases quickly degrade mRNA 
 

Initiation of  Can be inhibited by regulatory proteins that bind to specific sequences within 5’ UTR, preventing attachment of 
Translation   ribosomes 
 
Eg. Iron regulatory protein (IRP) binds at iron response element (IRE) (Refer to image)  

 
 
mRNAs are found in cytosol with long poly(A) tail (long enough for poly(A)-tail binding protein to bind but not enough 
to initiate translation) 
 
When protein is needed immediately, don’t have to wait for transcription and post-transcriptional control to happen. 
Instead, cytoplasmic enzymes will very quickly lengthen poly(A) tail to sufficient length to initiate translation. 
 
Translation of all mRNAs can be regulated simultaneously 
● Global control involves inactivation or activation of one or more protein factors required to initiate translation 
● Inactivation of eukaryotic initiation factors (eIF) 
○ Via phosphorylation  
● Activation of eIF 
○ After fertilisation, translation is triggered by sudden activation of translation initiation factors 
 Post-translational control 

Biochemical modification  Polypeptides must be processed to yield functional protein 

● Eg. Cleavage of initial insulin polypeptide forms the active hormone 
● Chemical modifications 
○ Phosphorylation (either activates or inactivates proteins) 
○ Glycosylation  
● Cell-surface and secretory proteins must be transported to target destinations in order to function 
● Regulation can occur at any step involved in modifying or transporting a protein  

Protein degradation   ● To mark a protein for destruction, cell attaches molecules of small protein called ubiquitin to target 
protein 
● Giant protein complexes called proteasomes recognise ubiquitin-tagged proteins and degrade them  

Gene Amplification 

Polymerase Chain Reaction  Step 1: Denaturation  

  ● Excess primer is mixed with DNA fragment 
Amplify specific DNA fragment  ● Thermal cycler heats mixture to 94°C 
● Hydrogen bonds holding double-stranded DNA fragment break and ds-DNA dissociates into 
single strands 
 
Step 2: Annealing of primers 
● Mixture cooled to 65°C 
● Due to large excess of forward and reverse primers, primers base paired with complementary 
sequence in single-stranded DNA, preventing them from reassociating back into double 
strands  
  
Step 3: Primer extension 
● Temperature raised to 72°C, which Taq polymerase functions best  
● Polymerase extends primer by catalysing phosphoester bonds in 5’ to 3’ direction 
● As both DNA strands are replicated, there are now two copies of the original fragment 
 
Single fragments after n cycle = 2n  

Gel Electrophoresis  ● DNA fragments are loaded into well of the agarose gel at the negative electrode 
  ● DNA fragments are negatively charged 
Separate DNA fragments according  ● When direct current is applied, electric field set up causes DNA fragments to move towards 
to size  the positive electrode 
● They are separated on the basis of size 
● Shorter DNA fragments move through the pores faster and form bands furthest away from 
the wells 

Southern Blot and Nucleic Acid  ● Restriction fragments 

Hybridisation  ○ Restriction enzyme is added to samples of DNA to produce restriction fragments 
  ● Gel electrophoresis 
Identify specific DNA sequence    ○ Separated by electrophoresis 
○ Each form a characteristic pattern of bands  
● DNA transfer 
○ Transferred by capillary action onto nitrocellulose membrane  
○ DNA is denatured using alkaline solution to form single-stranded DNA 
○ Single strands stick to membrane, positioned in bands exactly on gel  
● Hybridisation 
○ Blot is exposed to solution containing radioactively labelled single-stranded DNA 
complementary to DNA sequence of interest 
○ Attaches to restriction fragments with complementary sequence  
● Autoradiography 
○ Sheet of X-ray film laid over blot 
○ Radioactivity exposes film to form an image corresponding to position of gene of 
interest