Download Chapter 24 Genes and Chromosomes

Chapter 24
Genes and Chromosomes
Intro
Almost every cell contains DNA
DNA often packaged into structures called Chromosomes that contains a single
double strand of DNA
Bacteria and Viruses have a single chromosome
Eukaryotic cells usually many
Chromosome may contain thousands of genes
All of genes and intergenic DNA referred to a genome
For instance Yeast
16 chromosomes
range from 1.5x108 to 1x109 MW
that’s 230,000 to 1,532,000 bp
In B form chromosomal DNA is many orders of magnitude larger than cell itself
There must be lots of organization and tertiary packaging to make it all fit
In this chapter will
Look at size and organization of viral and cellular chromosomes
Look at Topology (coiling)
Finally DNA-Protein interactions
24.1 Chromosomal Elements
Cellular DNA contains both genes and regions between genes
Both may have functions in cell
also different levels of organization to watch out for
A. Genes are segments of DNA that code for polypeptide chains and RNA’s
Classical def of a gene: that portion of a chromosome that determines a
single character or phenotype (Mendelian genetics, Blues eyes, or black
hair)
Beadle and Tatum (1940's) proposed 1 gene- 1 enzyme model
Later refined to 1 gene - 1 protein
Modern def a little more specific, 1 gene 1 polypeptide
Even that’s not good enough because some genes code for RNA not
protein
Gene: the DNA that codes for the primary sequence of some gene
product
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DNA also has other information
Regulatory sequences
Where to start , where to stop, etc.
How much DNA?
3 base pairs/amino acid
Small peptide may be 50 AA (150 BP)
Average protein 350 AA (1050 bp)
Eukaryote and some prokaryote have noncoding DNA in middle to
make even longer
How many genes in a Chromosome?
Ecoli genome is a single chromosome that has now been
completely sequenced
4,639,675 bp
4,300 genes for proteins
157 genes for structural or catalytic RNA
Human Genome
3.1 billion base pairs
24 different chromosomes
25,000 genes
B. DNA molecules are much longer than the cell that contains them
Viruses
Not a free-living organism, but an infection parasite
Can contain just DNA(or RNA) and a protein coat
Sizes vary
See table 24-1
During replication physical form of DNA genetic material may
change
Single strand becomes double strand
Linear become circular
RNA is replicated into DNA
In all cases contour length of DNA is larger than viral particle
Bacteria
Roughly 10x larger than viruses
E coli
4,639,675bp. It has been sequenced)
About 4,300 proteins and 157 RNA
Length 1.7 mm (~850x length of cell)
Many bacteria also contain plasmids
Extrachromosomal DNA
Usually 1,000- 10,000 bp
Self replicating DNA
Usually no advantage for host
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But sometimes can carry an antibiotic resistance
gene
Leftover virus like DNA? Leftover sexual
reproduction?
Can be transmitted from one bacteria to another of same
type
If carries a gene for antibiotic resistance, can help a
create a antibiotic resistant bacterial strain
Eukaryotes
Simplest 2.6x more DNA than bacteria
Fruit fly 35 x as much DNA
Human 700 x as much DNA
Often diploid (2 copies each chromosome)
Each chromosome = 1 DNA molecule
Human 46 chromosomes
22 matched pair
X and/or Y
Length varies - some as small as 1/25 of largest
Each cell ~ 2m of DNA
1014 cells / body
1 body contains 2x1011km DNA
Distance from earth to sun 1.5x108 km!
3,200,000,000 bp
Nature 431 931(2004) estimate 20,000-25,000
Also don’t forget DNA of mitochondria or chloroplasts
Human mitochondria 16,569 bp
Each mito has 2-10 copies (can be up to thousands)
C. Eukaryotic Chromosomes are very complex
Bacteria
Usually 1 chromosome
Usually 1 copy of each gene, although some RNAs have
several copies
Regulatory sequences and genes account for most of DNA
Gene corresponds almost exactly to sequence of protein
Eukaryotic much more complex structurally and functionally
Introns
Most eukaryotic genes contain Introns
Intervening sequences that do NOT code for peptide product
Intervening sequences - introns
Expressed sequences - exons
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Very few introns in prokaryotes
Amount and size of introns vary from gene to gene
(See figure 24-7)
Some proteins, (histones) have no introns
Function of introns is not clear
Only 1.5 % human genome is exons!
Introns and exons account for 30% of genes
What is the rest?
45% Transposons or transposon derivatives
More details in later chapters
Transposon -Transposable elements
(Can move from one location to another in the
genome)
100's to 1,000 of bp
Generally don’t encode for anything useful
3% highly repetitive DNA
Also called simple-sequence DNA
Also called simple sequence repeats (SSR’s)
Also called satellite DNA
Sequences about 10 bp long, repeated up to a
millions times
Does have useful function
Centromeres & telomeres
(Figure 24-8 - on board)
Centromere
Where chromosome is linked to mitotic spindle
To separate chromosomes during cell division
Typically 1000's of tandem copies of a few
short 5-10 bp sequences
Function not yet understood
Telomeres
Stabilized end of DNA
100 bp of imprecisely repeated
(5') (TxGy)n
(3') (AxCy)n
X&Y between = 1-4
N= 20-100 single cell eukaryots
N= 1,500 mammals
Used to replicate ends, since DNA is not
circular
More details in later chapters
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24.2 DNA Supercoiling
have seen over and over evidence that DNA must be compacted
and must be able accompanied in replication and transcription
One more important property - Supercoiling
Coils of coils
Telephone chord
regular coils
and supercoils (figure 24-9, but bring one in from home or rip off of office
phone before class)
DNA - regular coiling is the 10.5 bp/turn coiling
supercoiling is any bending of DNA helix itself
No supercoiling - DNA said to be relaxed
Supercoiling occurs in all cells and is highly regulated by cell
can be studied mathematically using topology
A. Most cellular DNA is underwound
Start with small circular DNA’s (viral or plasmid)
If no breaks in either strand - called closed circular DNA (ccDNA)
If ccDNA is relaxed, then in B form 10.5 bp/turn
When isolated from cell almost never relaxed
So cell has induced supercoiling in DNA
Almost always DNA is underwound
I.e. has fewer helical turns that B-form DNA
Say had 84 bp of DNA
Expect 84/10.5 = 8, or DNA to have twisted around itself 8 times in
making the cc DNA
If removed one turn would have 84/7 or 12 bp/turn
Since this is not thermodynamically stable the DNA secondary
structure will stay at 10.5 bp/turn, but one loop will pop into tertiary
structure
See figure 24-13
All cells have underwound DNA
Thought to be for two reasons
1. If underwound, then as saw above, DNA makes supercoils, and
this helps compact the DNA.
2. Also as shown in figure 24-13, if the underwinding of the DNA
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can be localized to a single region, it is like having this region with
the DNA separated, so underwinding DNA makes it easier to start
strand separation helps the binding of proteins like histones
B. DNA underwinding defined by topological linking number
Now let’s look at the math
Linking number (Lk) math property, invariant as change structure as long
are remains cc
Mathematically lk is the number of times the second strand pierces the
surface defined by the first strand
Figure 24-14
If Lk =1 cannot separate
If Lk=6 you start to see the helical structure
Lk # for ccDNA always integers
for right handed helices lk is defined at +
B DNA is right handed so see + Lk in biology
If have 2,100 bp ccDNA phasmid relaxed, 10.5 bp/turn
2100/10.5 , Lk=200
Now as long as remains cc, Lk # is set, now matter how we
manipulate DNA
Now for a little math
Say do use some process to change the Lk number to 198
ÄLk = 200-198 = -2
And the DNA now has 2100/198 or 10.6 bp/turn
Since secondary structure of DNA really wants to have 10.5 bp/turn
you will get -2 supertwists in the structure
Have a number called ‘specific linking difference’ (ó) or
‘superhelical density’ to express # of supertwists/ preferred link
number
Where Lko is the preferred Lk # of a piece of DNA
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For our example = -2/200 = -.01
Typical cellular DNA is 5-7% underwound
Or ó = -.05-0.07
So DNA usually has LESS turns than it wants
Called negative supercoiling
Positive supercoiling can exist but usually in the lab, not in the cell
DNA molecules that differ by Lk # are called topoisomers
Lk number has two components, the writhe (Wr) and the twist (Tw)
Lk=Tw+Wr
Describing writhe and twist more difficult (figure 24-17)
Twist is local twisting. Corresponding to bp/turn
Writhe is global twisting, correspond to the supercoiling
twists we have seen
Tw and Wr are defined by local geometry and can have nonintegral values
C. Topoisomerases catalyze change in linking number
enzymes that change linking # in DNA are called topoisomerases
play a special role in DNA replication and packaging
2 main types
Type I - transiently break 1 strand, and rotate other strand around
change in Lk in steps of 1 (Mech figure 24-20)
Type II - Transiently break both strands changes Lk in steps of 2
(Mech figure 24-21)
Passes one entire strand through another
Typically takes ATP
Can see in agarose gels Figure 24-19
As change Lk, change supercoils, and DNA runs at a different
speed
4 topoisomerases identified in E coli
I and III are type I and relax DNA by removing negative supercoils
II also called DNA gyrase uses ATP to put negative supercoils into
DNA using type II mechanism
Both types of topoisomerase also observed in mammalian cells, but lets
not get into the confusing nomenclature. One important point mammalian systems do NOT have an enzyme that can introduce negative
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supercoils! Yet, in its native form, mammalian DNA has negative
supercoils!! Will explain in minute
D. DNA Compaction requires special form of supercoiling
Supercoiling in DNA can have 2 different forms
Plectonemic =twisted thread
Solenoidal = made into little coils
See figure 24-23
Plectonemic is what is observed in naked DNA (figure 24-22)
It reduces the length of DNA a bit, but not orders of
magnitude
Solenodial on the other hand accomplished great compaction of
DNA
Not found in naked DNA
Both forms can occur in theory and readily interconvert. However
naked DNA is usually Plectonemic, and DNA is not seen in
Solenoidal form unless you add protein Can you guess why??
(Solenoids, lots of negative charge would repel and keep from
collapsing. Protein can supply + charge to compensate
Solenoid form is key to chromosome structure
24.3 The Structure of Chromosomes
Have seen chromosome applied to DNA molecule that stores the genetic code of
a cell
Term also used for densely colored bodies seen in the stained nuclei of
eukaryotic cells
Sharply defined structure observed just before and just after mitosis (nuclear
division)
A. Chromatin contains DNA and protein
(Figure 24-24)
In non-dividing eukariotc cells , in phase G0
And in interphase (G1,S,G2)
Chromosomal material, called chromatin
Is amorphous, and seems to be randomly distributed in nucleus
In S phase DNA is replicating and making 2 chromosomes for
every one at start, but chromosomes are still associated
During prophase of Mitosis chromosomes become much more condensed
This is stage where is visible under light microscope Figure 24-5
Andi it is these bodies that are referred to as chromosomes
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Chromatin - roughly equal amounts of protein and DNA and a touch of
RNA
DNA closely associated with protein called histones and together make a
structure called the nucleosome (see figure 24-25)
Chromatin also contains many nonhistone proteins some of which are
regulatory in nature, others are used to manipulate the DNA
The nucleosome is the first layer in several of packaging that take DNA
and turns it into a visible chromosome. Will now look at structure, and
also do some comparison with prokaryote packaging as well.
B. Histones are small basic proteins
Table 24-4 and figure 24-26
histone proteins vary from 11,000-21,000 MW rich in Arg and Lys(2030%)
5 classes
H1, H2A, H2B, H3,H4
H3 and H4 nearly identical in sequence for all eukaryote.
Suggests structure and function strongly conserved
H1, H2A and H2B not as closely related
Several post-translational modifications observed
ADP-ribosylatin, phosphorylation, and acetylation
Affect physical structure
Plays a role in regulation of transcription (Chapter 28)
C. Nucleosomes are Fundamental organization unit of Chromatin
In packing DNA into a chromosome typically start with 105 ìm of DNA and
shortens it down to 5-10 ìm (105ìm=102mm = 100mm=.1m)
So .1m shortened to .005 mm
Lots of levels of structural compaction lowest has to do with histones
at the lowest level you can observe DNA that look like “beads on a string”
(figure 24-25 again)
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The nucleosome consists of the bead and the DNA spacer to the next
bead.
Each nucleosome bead has 8 histone proteins
2 each of H2A,H2B, H3, and H4
Bead spacing about 200 bp/nucleosome
146 bp are wrapped around the histone core in two turns
Remaining 56 is in the linker region
Histone H1 binds to the linker DNA
Also required for 30 nm fiber on next page
Extending out from core are amino terminal tails of histones
24-26 d & e
Tails are intrinsically disordered
Tails are where most of the modifications occur
End up being key in contact needed for higher order
structure
When DNA wrapped around the nucleosome get a solenoidal supercoil
that change in Wr that should introduce a + supercoil into the unbound
DNA (figure 24-27) DNA wraps around histone in left hand manner so get
- supercoil
Now lets return to an earlier problems I mentioned. How can mammalian
DNA be negatively supertwisted, when mammalian cells have no
enzymes to introduce supertwists?
Let’s go back to our figure with DNA negatively supertwisted onto a
nucleosome, with a compensating supertwist on the non-bound DNA
What happens if one or our regular topoisomerases comes and
relaxes the unbound DNA? (Figure 24-27 part C)
And now what happens when we release the DNA from the
histone?
Behold, negative supercoils,
More details
Location on histone on DNA is not random
Not well understood
Seem to prefer to bind to AT base pairs in minor groove
(Figure 24-28)
Thought to be tied to fact that is easier to unravel DNA
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around AT pairs
In several organisms protein discovered that bind to a specific
sequence of DNA and nucleate formation of nucleosome core
nearby
Location of cores can play a role in gene expression
We now know some details of histone assembly
Required when first making chromatin, and during disassembly
/reassembly during transcription
Tetramer of 2-H3's and 2-H4's bind first
Then H2A-H2B dimers
After chromosomal replication this process requires
‘Histone chaperones’
Chromatin assembly factor 1 (CAF-1)
RTT106
Antisilencing factor 1 (ASF-1)
Bind to acetylated H3 and H4
But the rest is still under investigation
(And has changed since 5th edition)
D. Nucleosomes are packaged into higher order structures
nucleosome packing give you about a 7 fold compaction, need about
10,000 fold
Depending on how gently you are you can get Eukaryotic DNA in a 30 nm
fiber (~ 100 fold compaction)
Structure shown in figure 24-29
required 1 histone H1 for each nucleosome
not uniform for all DNA. Non-histone binding protein will bind to DNA and
break fiber structure
Not observed in DNA being transcribed
Transcribed DNA less-ordered and little or no H1
Higher orders of compaction not well understood
Figure 24-31
30 nm fibers supercoil and associate with nuclear scaffold
Loops of DNA 20,000-100,000 bp
Again not random
Loops seems to be related genes
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Scaffold is protein seem rich in H1 protein and topoisomerase II
If use inhibition to stop topoII can kill rapidly dividing cell
Used as a cancer chemotherapy
Several other layers of structure
No complete description at this time
E. Condensed structure maintained by SMC proteins
SMC- Structural Maintenanceof Chromosome
Found in all organisms from bacteria to people
Figure 24-32
5 domain protein
N domain
Helical coiled-coil
Hinge
Helical coiled-coil
C-domain
Coiled coils - coil around each other
Bring N and C domains together
In fact need together to get ATPase site
works as a dimer
Joined at the hinges
Makes a V-shaped molecule
Eukariotes - SMA proteins further subdivided into 2 types
(figure 24-32c)
Cohesins - link together sister chromatids as chromosomes
condense in metaphase
So important in Mitosis itself
Condensins - Condense DNA as enter mitosis, so important in late
stage of interphase
In lab condensins introduce + supercoils as DNA is bound
Not sure why or how
Also a third protein - kleisin
Used to link 1 arm of V to other to from ring around replicated DNA
Finish with figure 24-34
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F. Bacterial DNA also organized
in bacteria have a structure called the nucleoid
See figure 24-35
while not a membrane bound organelle can see DNA localized to a
distinct region
DNA attached at one or more points to PM
Circular DNA organized into a series of looped domains
Figure 24-36,
500 looped domains, each contains ~10,000 bp
Can tell each domains is topologically constrained
Can nick in 1 domain and not relax another
But move as DNA is replicated and trascribed
Are histone like proteins but bind and dissociate in a matter of minutes
So not stable histone like structure
Dynamic structure of bacterial chromosome simply reflect a much more
dynamic cell
Can replicate in as little at 15 minutes
Much more of DNA is used to encode for proteins products
So need to have access to this DNA continually