Genome organization in eukaryotes

• protein coding regions are rare

(~1.5% in humans)
• genes are scattered throughout the
genome
• flanked and interrupted by
non-coding DNA
• coding regions are conserved across
(related) species, but non-coding
regions are not
• many regions of different sizes are
duplicated

Alberts (2015) Molecular Biology

of the cell. 6th edition, Figure 4-15
 Genome organization: non-coding DNA
• the vast majority of eukaryotic genomes has no known function!
• unique sequences
• introns
• non-repeated, non-coding
intergenic DNA (including
cis-regulatory elements)
• repetitive sequences
• long, but low copy (duplications of
parts of the genome [segmental])
• short, but many local repeats Alberts (2015) Molecular Biology
(microsatellites = simple sequence repeats) of the cell. 6th edition, Figure 4-62

• intermediate in length, many copies scattered through genome:

• (Remnants of) Mobile DNA elements (Transposons)
 Genome organization: non-coding DNA
• the vast majority of eukaryotic genomes has no known function!
But is it actually functionless?

• 80% of the bases of the human

genome are:
• transcribed (75%)
• associated with modified histones (56%)
• found in open-chromatin areas (15.2%)
• binding transcription factors (8.5%)
series of papers published in 2012:
à 80% of the genome is functional (ENCODE) http://www.nature.com/encode/
https://www.encodeproject.org
(but, is this a valid conclusion?)
 Mobile DNA Elements (Transposons)

The original idea:

all loci are placed in the genome in a linear and stable manner
à necessary to explain classical (Mendelian) inheritance

The modification:
most loci are placed in the genome in a linear and stable manner
BUT some loci can excise themselves and re-integrate into a different genome
location
à called ‘jumping genes’, ‘transposons’ or ‘mobile elements’
 Genome organization: non-coding DNA

• genetic elements that can move from one place in the genome to another
• excision/ reintegration can lead to duplication of transposons
• occur in very large copy numbers (300,000 to 1.600,000 in humans)
• can make up large fractions in genomes
• do not have an immediate physiological function
• considered to be ‘endosymbionts’
or ‘endo-parasites’
à ‘selfish genes’ or ‘junk DNA’

But: have huge implications

in genome evolution!!!
 Classes of Mobile DNA Elements
Major classes of transposons
1) DNA transposon 2) retro-transposon
• two major categories
• two major categories
1. transpose as DNA former
donor DNA
flanking
donor DNA

DNA RNA
transposon
1. transpose as (DNA
DNA transposons)
(DNA transposons) site
polymerase

Transposase
• • involves transposase
involves transposase RNA intermediate
donor DNA
Reverse
DNA
• cut and paste• mechanism
cut and paste mechanism intermediate
transcriptase

2. transpose via RNA intermediate

(retro-transposons)
2. transpose via RNA intermediate Transposase target DNA Integrase

(retro-transposons)
• involves reverse transcriptase

• involves reverse
• andtranscriptase andto
integrase (similar
transposase)
integrase (similar to transposase) transposed
mobile element
• copy
• copy and paste and paste mechanism
mechanism
also see: Table 5-4 in Lodish et al. (2003) Molecular Cell Biology,
Alberts, 6th edition Fith Edition Freeman Pub.: Chapter 10.3,
Figure 10-8
 Discovery of transposons

• studies on two maize (corn) mutants in 1940s by Barbara McClintock:

• gene C: necessary for kernel colour formation
• c/c = white kernels (mutant)
• C/c or C/C = purple kernels (WT)

• gene Ac (Activator):
• if Ac (a dominant allele) is present in c/c:

icpage/barbara-mcclintock-and-the-
https://www.nature.com/scitable/top
Pray et al. (2008) Nat. Edu. 1:
• high frequency of revertants back to C/c

discovery-of-jumping-34083
• revertant can affect whole kernel or only sectors
(the earlier in development reversion happens,
the larger the WT sector)
 Discovery of transposons
• genetic mapping and phenotype analyses (by McClintock):
• mutation in C-gene caused by integration of another genetic element (called Ds)
• reversion is caused by dissociation of Ds (Dissociator) from C-locus
• Ds no longer genetically linked to C-locus in revertants!
• ‘dissociation” leads to mapping of Ds to another
chromosomal locus
• Ac activates re-location (or transposition) of Ds
• the Ac locus maps to different location than C
(and can change position as well…)
à both Ds and Ac can change location in genome
à transposition of Ds causes mutation / reversion of C-gene
à Ds can only move if Ac is present
 The Ac / Ds system

• Both Ac and Ds are DNA transposons

is a functional DNA-transposon
• Ac element is autonomous
• Ds element inisnon-autonomous
a internally truncated version
• Ac encoded transposase mediates
transposition of itself and of Ds
encoded transposase mediates
• random integration into C caused “white kernel”
phenotype (rare event)
• excision of Ds reverts disrupted C-gene
back to normalexcision does not leave copies or
• excision of Ds during kernel development causes
reversion to WTexcision (fairly in
phenotype frequent)
affectedofsectors
Ds during
(happens everykernel
time development
it moves) causes reversion
to WT phenotype in affected sectors
 Barbara McClintock and the Noble Price

• Discovered jumping genes and transposition in the 1940’s and 50’s.

• Received Nobel Price only in 1983. Why did it take so long?
• Peer discrimination: she was very eccentric and a women
• Existence of transposition was accepted, but seemed
limited to maize (until 1970’s: bacterial transposons)
• Biological significance and spread only fully appreciated, when
large-scale sequencing began
• Main reason: she miss-interpreted her results as a regulatory
mechanism, not as a random process
• Ds integration into C: a repressor of gene C activity
Comfort (2001) TiBS 26: 454-457

• Ac: a repressor of the repressor (ie activator of gene C)

 McClintock’s hypothesis about transposon function
• gene control mechanism (analogous to lac-operon regulation)
• if Ds located away from target gene: no repression
• Ac activates Ds translocation into target gene: repression
• Ds translocation reversible by Ac action: (dissociation) --> no repression

this hypothesis
turned out to be
wrong!

Comfort (2001) TiBS 26: 454-457

 Structure of DNA transposons

• occur in both prokaryotes and eukaryotes

• similar structure and mode of transposition
• Examples: Ac/Ds in maize,
P-element in Drosophila,
IS element in E. coli
 Jumping of DNA transposons
Alberts (2015) Molecular
1. Transposase Biology of the cell. 6th
edition, Figure 5-61

• cuts donor DNA blunt at

ends of inverted repeats
• cuts target DNA staggered
creating overhangs
2. Transposase
• ligates transposon into target site
3. Cellular DNA polymerase and ligase
• fill in overhangs and join ends
à creates short direct repeats specific for
target site
à leaves 9 bp duplication when excised again
 Amplification mechanism of DNA transposons

• Mode of jumping (copy-

paste) does not per se lead
to increase in transposon
copies
• Amplification can only
occur during replication of
host DNA, if:
• transposon jumps from
already replicated DNA
to a region not yet
replicated, and
• transposon jumps in a
germ line cell
 Summary: DNA transposons
• The Ac/Ds system affecting colour development in maize were the first transposons discovered
• Transposition occurs via a cut-paste mechanism
• Inverted repeats on each site of the transposon act in cis:
• their 5’-end is recognition site of transposase, which cuts them blunt end
• transposase is encoded in between inverted repeats
(at least in completely functional [autonomous] transposons)
• Expression level of transposase gene defines frequency of transposition
• may be developmentally regulated or limited to low levels
• may act on own transposon or in on other transposons
• Transposase cuts randomly in genomic DNA creating sticky ends
• Transposase also ligates excised transposon into cleaved target site
• Amplification of element ONLY occurs during replication
 Classes of Mobile DNA Elements
Major classes of transposons
1) DNA transposon 2) retro-transposon
• two major categories
• two major categories
1. transpose as DNA former
donor DNA
flanking
donor DNA

DNA RNA
transposon
1. transpose as (DNA
DNA transposons)
(DNA transposons) site
polymerase

Transposase
• • involves transposase
involves transposase RNA intermediate
donor DNA
Reverse
DNA
• cut and paste• mechanism
cut and paste mechanism intermediate
transcriptase

2. transpose via RNA intermediate

(retro-transposons)
2. transpose via RNA intermediate Transposase target DNA Integrase

(retro-transposons)
• involves reverse transcriptase

• involves reverse
• andtranscriptase andto
integrase (similar
transposase)
integrase (similar to transposase) transposed
mobile element
• copy
• copy and paste and paste mechanism
mechanism
also see: Table 5-4 in Lodish et al. (2003) Molecular Cell Biology,
Alberts, 6th edition Fith Edition Freeman Pub.: Chapter 10.3,
Figure 10-8
 Retrotransposons

• two major classes

1. retroviral-like retrotransposons (contain long direct terminal repeats, LTR)

2. non-retroviral like retrotransposons (contain AT-rich regions at flanking sites, non LTR)

Alberts (2015) Molecular Biology

of the cell. 6th edition, Table 5-62
 Transposon
• indeed, retroviruses likely evolved evolution
from transposon:

transposase (Trp)
evolved to integrase
predecessor acquired
reverse transcriptase
(RT) and RnaseH (RH)

acquired LTR’s
and a protease
(processes primary
protein to RT and RH)
from: Biémont and Vieira
(2006) Nature 443: 521-524

acquired envelope proteins

(allow leaving the host cell)
 Life cycle of retroviruses and LTR transposons
• LTR-transposon and retro-
integrated DNA
viruses share mechanism of integrase
movement / amplification modified from Alberts (2015)
Molecular Biology of the cell. 6th
RH and RT
• RNA and proteins made by edition, Figure 5-62

regular cellular transcription

/ translation machinery
RT transcription assembled new viruses
• viruses only have envelope (including enzymes)
proteins
• allows movement from
cell to cell

retrotransposons
matrix / capsid

retroviruses
• retrotransposons only proteins
cell entry and
form matrix loss of envelope envelope proteins

• allows amplification
enzymes
within cell only (RT, RH, integrase)
 Life cycle of retroviruses and LTR transposons
• reverse transcriptase (RT) is
a DNA polymerase that can
use RNA or DNA as a
template
• RNaseH (RH) degrades RNA
if in RNA/DNA duplex
integrated DNA
integrase
modified from Alberts (2015)
RH and RT Molecular Biology of the cell. 6th
edition, Figure 5-62
RT transcription assembled new viruses
(including enzymes)

• integrase acts similar to

transposase of DNA
transposons

retrotransposons
matrix / capsid

retroviruses
proteins
cell entry and
loss of envelope envelope proteins

enzymes
(RT, RH, integrase)
 The LTR problem
The LTR Problem
• LTR contains promoter driving transcription (the U3 part):
• LTR contains promoter driving transcription (the U3 part):

integrated retroviral or
LTR retrotransposon DNA

• RNA intermediate lacks the

promoter (the U3 part of
primary transcript the LTR) at 5’ end and the
U5 part at the 3’end
• How are they recovered?
Lodish et al. (2003)
Figure 10-12
retroviral or retro-
transposon RNA genome R U5 U3 R

• RNA intermediate lacks the promoter (the U3 part of the LTR) at 5’ end
The LTR solution
 Back to Overview

• two major classes

1. retroviral-like retrotransposons (contain long direct terminal repeats, LTR)

2. non-retroviral like retrotransposons (contain AT-rich regions at flanking sites, non LTR)

Alberts (2015) Molecular Biology

of the cell. 6th edition, Table 5-62
 Non-LTR transposons
• most abundant types in mammals:
• long interspersed elements (LINE): ~6kb
• ~900,000 copies in human genome
• but only ~0.01% are full length
• short interspersed elements (SINE): ~0.3kb
• ~1,600,000 copies in human genome (~13% of total genomic DNA!)
• contain no coding regions but only flanking regions (of LINES)
• use the LINE encoded machinery to amplify
• expressed from
• promoters outside the actual transposon (depend on integration close to promoter)
• or from internal promoter (SINE)
• scattered throughout the genome, but hugely concentrated in a few genomic regions
 Non-LTR transposons:
movement and amplification

• only autonomous transposon encode

Reverse transcriptase / endonuclease
• enzyme can act both on it’s own and
other transposons of the same class
• reverse transcription and integration are
coupled
• movement always leads to increase in
transposon copy number

Alberts (2015) Molecular Biology

of the cell. 6th edition, Figure 5-63
Multistep pathway during non-LTR transposon movement
 Summary transposons
• DNA transposons ‘jump’ via a cut-and-paste mechanism
• amplification can only occur during replication
• RNA transposons amplify via transcription and reverse transcription (copy-paste)
• LTR retro-transposons can be completely autonomous
• transcription starts within transposon
• must regenerate transcription start site
• require reverse transcriptase (RT), RNaseH, and integrase
• evolved to retroviruses (allows ‘jumping’ into genomes of other cells)
• non-LTR retro-transposons
• most rely on transcription from host promoters
• require RT only