Genes and Chromosomes

Key topics:
Organization of information in chromosomes
DNA supercoiling
Structure of the chromosome
Understanding of Genes Has Evolved

Historically: a gene was something responsible

for a characteristic or phenotype
Beadle and Tatum, through mutation studies,
defined a gene as something that coded for an
enzyme (one gene one enzyme)
Now: genes defined as segments of DNA that
code for peptides and RNA
Different from regulatory sequences
DNA can be expressed differently to yield different
products (one gene different products)
DNA is a very large macromolecule

The linear dimensions of DNA are much bigger than

the virions or cells that contain them
Bacteriophage T4: ~0.2 m long and 0.1 m wide
T4 DNA: ~60 m long
So DNA in a virion or cell is organized into compact
forms, typically via coiling and association with
proteins
Linearized DNA from Bacteriophage T2
 Molecular Coding of Protein
Sequence Information
In transcription, one strand of double-stranded DNA
acts as the molecular template for RNA synthesis
(DNA messenger RNA)
3 nucleotide codons provide information
In translation, the triplets of nucleotides in mRNA bind
to complementary triplets in tRNA
The tRNA molecules contain amino acids associated with the
particular triplet
Amino acids are thus assembled in peptides
Protein sequence determines its biological function
 How many genes per organism?

E. coli (single circular chromosome) 4,639,675 bp ~4300 genes

for protein, ~157 for catalytic or structural RNAs

Human (23 discrete chromosomes) 3.2 billion bp 25,000 genes

Many viral genomes are RNA

Many viruses: only RNA or DNA surrounded by

protein coat
They use hosts genes when they infect plant, animal or
bacterium
RNA genomes may be small and single-stranded
HIV: 9000 bp
Phage Q: 4220 bp
Genomes may change form from circular to linear,
etc. during life cycle
Bacterial genomes are double-stranded circles
E.coli: 4,639,675 bp
850 longer than the cell!
Bacteria also contain extra-chromosomal, double-stranded
circular plasmids
~200010,000 bp
Swapped easily between bacteria
No essential genes but often encode genes that degrade
antibiotics
Plasmid exchange is one way bacteria acquire antibiotic resistance
Length of E. coli DNA Relative to
Length of Cell
DNA from Lysed E. coli Cell
 Eukaryote DNA is in multiple discrete
chromosomes

Number varies with species

Human somatic (non-sex) cells have 46 chromosomes
22 pairs (diploid) plus X and Y
Amounts to ~2 m of DNA
Length varies
Eukaryotic
Chromosome
Mitochondria and chloroplasts also
have DNA

Double-stranded circles
Human mitochondrial DNA (mtDNA)
16,569 bp
~210 mtDNAs per mitochondrion
Plant mtDNA
200,0002,500,000 bp
Chloroplast DNA
120,000160,000 bp
Mitochondrion
DNA, Chromosomes, Genes, and
Complexity
Neither the total length of DNA, nor the number of
chromosomes correlates strongly with the complexity of an
organism
Dogs and coyotes have 78 chromosomes
Amphibians have much more DNA than humans
Plants have more genes than humans
The correlation between genome size and complexity is poor
because most of eukaryotic DNA is non-coding

Recent experimental work by Craig Venter suggests that a living

organism could get by with less than 400 genes
DNA is packaged with proteins
Viral genomic DNA may be associated with
capsid proteins
Prokaryotic DNA is associated with proteins
in the nucleoid
Eukaryotic DNA is organized with proteins
into a complex called chromatin
Composition of the Human Genome
Notice that only a small fraction (1.5%) of the total
genome encodes for proteins
The biological significance of non-coding sequences
is not entirely clear
Some DNA regions directly participate in the regulation
of gene expression (promoters, termination signals, etc.)
Some DNA encodes for small regulatory RNA with
poorly understood functions
Some DNA may be junk (pieces of unwanted genes,
remnants of viral infections
Eukaryotic genes contain intervening
sequences (introns)
Exons are expressed sequences (translated into
amino acid sequence)
Exons account for only 1.5% of human DNA!
Introns are regions of genes that are transcribed
but not translated
They do not encode polypeptide sequence
Introns are removed after transcription and the
exon mRNA sequences are spliced together
creates mature transcripts
Introns in Two Eukaryotic Genes
 Some bacterial genomes also contain
introns
Until 1993, scientists thought that introns appeared only in eukaryotes
About 25% of sequenced bacterial genomes show presence of introns
Introns in bacterial chromosome do not interrupt protein-coding
sequences; they interrupt mainly tRNA sequences
Introns in phage genomes within bacteria interrupt protein-coding
sequences
Many bacterial introns encode catalytic RNA sequences that have
ability to insert and reverse transcribe themselves into the genomic
DNA
Transposons are sequences that can
move within the genome
Eukaryotic genome is not completely static
Sequences called transposons can move around within
the genome of a single cell
The ends of transposons contain terminal repeats
These repeats hybridize with complementary regions
of target DNA during insertion
Transposons account for ~50% of human genome
Eukaryotes also contain highly repetitive DNA
or simple sequence repeats (SSRs)

Short sequences ~10 bp or less

Repeated millions of times
Also known as satellite DNA
because when fragmented and centrifuged, the DNA
separates into a discrete satellite band
Associated with centromeres and telomeres (next
slides)
 Telomere sequences cap the ends of
eukaryotic chromosomes

May form special loop structures to keep DNA ends

from unraveling
Contain multiple repeats with general sequence
(TxGy)n
(AxCy)n where n ~ 1500 in mammals

Added by enzyme telomerase

 Telomeres are associated with cellular
aging

In many tissues, telomeres are shortened after each round

of replication
Thus, the cellular DNA ages
Normal human cells divide about 52 times before losing
the ability to divide again (Hayflick limit)
Centromere sequences are where proteins
attach during mitosis

Region where the two daughter chromosomes are

held together during mitosis
that is, after DNA replication but before cell division
Essential for equal distribution of chromosome
sets to daughter cells
Have AT-rich, repeated sequences of ~130 bp
Telomeres and Centromeres in a Yeast
Chromosome
DNA Supercoiling

DNA in the cell must be organized to allow:

Packing of large DNA molecules within the cells
Access of proteins to read the information in DNA
sequence

There are several levels of organization, one of which

is the supercoiling of the double-stranded DNA helix
Helical Supercoils
 Supercoiling is the coiling of a coil

Non-supercoiled DNA is called relaxed

Many circular DNAs are supercoiled
Supercoiling has great influence on transcription and
replication of DNA
Supercoiling can be highly regulated
Closing DNA in a loop introduces supercoiling
The Effects of Replication and Transcription on
DNA Supercoiling
Most cellular DNA is underwound

Normal B-form, relaxed DNA: 10.5 bp/turn

Closed circular DNA is rarely relaxed
Strain induces supercoiling
Strain is due to fewer helical turns (underwinding)
Underwinding makes later separation of the strands
easier
Linear DNA is underwound with the help of
proteins to prevent strands from rotating
Relaxed and Increasingly Supercoiled
Plasmid DNA
Effects of DNA Underwinding
Linking number (Lk) describes supercoiling

In circular DNA, changing the helical turns requires

breaking a strand transiently
Linking number in relaxed DNA:
Lk = #bp #bp/turn

Example: relaxed circular dsDNA of 2100 bp in the B

form (10.5 bp/turn) has
Lk = 2100 bp 10.5/turn = 200

Lk is an integer for closed-circular DNA and is (+),

reflecting a right-handed helix.
Lk is the number of times a strand
penetrates a surface
Lk Applied to Closed-Circular DNA
 Changes in Lk are useful for
describing supercoiling
Consider a relaxed DNA of 2100 bp:
Lk0 = 200

Underwind by removing two helical turns:

Lk = 198
Lk = 198 200 = 2
Superhelical Density,
Negative and Positive Supercoils
 Linking number can be broken down into
Twist (Tw) and Write (Wr)
Lk = Tw + Wr
Twist (Tw) is the # twists or turns of the helix
Writhe (Wr) is the # coils
Typically a negative value
 Underwinding Facilitates Additional DNA
Structural Changes

Helps to maintain structure of cruciforms at palindromes (next slide)

Cruciforms rarely occur in relaxed DNA

Facilitates formation of stretches of left-handed Z-form

Cruciforms
 Topoisomers are DNAs that differ only
in linking number

Same # bp, same sequence but different degree of

supercoiling

Conversion between topoisomers requires a DNA

strand break

Note that negatively supercoiled DNA (more compact)

travels faster in agarose gel electrophoresis experiment
than relaxed or nicked DNA
Topoisomers in Electrophoresis
 Topoisomerases are enzymes that change Lk

These enzymes are required for DNA unwinding and

rewinding during transcription and replication
Two major types:
Type I make a transient cut in one DNA strand
Type II make a transient cut in both DNA strands, change
Lk in steps of 2.
 The Topoisomerases IIV of
E. coli
Topo I and III are Type I
removes negative supercoils to relax DNA
Use single-strand breaks
Topo II is called DNA gyrase
Introduces negative supercoils
Uses ATP and double-strand breaks
Type 1 Topoisomerase
Eukaryotic topoisomerases include
Topo I, II, II, IV
Topo I and III are Type I (as in E. coli)
Type II topoisomerases include two subfamilies
Type IIA and Type IIB
Can relax both positive and negative supercoils
Mechanism of a Eukaryotic
Type IIA Topoisomerase
Topoisomerases are targets for
antibiotics

Coumarins (novobiocin, coumermycin A1)

inhibit bacterial type II topoisomerases from binding
ATP

Quinolones (nalidixic acid; ciproflaoxadin, Cipro)

Inhibit the last step, which is resealing the DNA strand
breaks
Wide-spectrum and mostly selective for bacterial
enzymes
Topoisomerase Inhibitors Used as
Antibiotics
Topoisomerase Inhibitors Used as
Chemotherapy Agents

Targets cancer because most rapidly growing cells

(tumors, others) express topoisomerases
Eukaryotic Type I topoisomerase inhibitors
Captothecin, irinotecan (Campto), topotecan
(Hycamtin)
Trap the enzyme-DNA complex in its cleaved state
Eukaryotic Type II topoisomerase inhibitors
Doxorubicin (Adriamycin), etoposide (Etopophos),
ellepticine
Topoisomerase Inhibitors Used as
Chemotherapy Drugs
 Supercoiled DNA forms Plectonomic or
Toroid/Solenoid structures
(or a combination)
Plectonomic
Seen in plasmids
Involves a right-handed superhelix with terminal loops
Toroid/Solenoid
Used in chromatin
Involves tight left-handed turns
Can resemble a garden hose on a reel
Provides more compaction
Plectonemic Supercoiling
Plectonomic and Solenoidal
Supercoiling
 Eukaryotic chromosome structure
changes over the course of a cell cycle
Nondividing state (G0) and Interphase (Gap1, G1; Synthesis, S; and
Gap2, G2):
Chromatin is amorphous, randomly dispersed
Prophase of mitosis
Chromosomes become condensed, pairs of sister chromatids form
DNA Packing into Chromatin
Chromatin consists of fibers of protein and DNA
and small amount of RNA
DNA associates tightly with proteins called
histones
Small proteins with lots of basic (Lys, Arg)
residues
Are modified by methylation, acetylation, ADP-
ribosylation, phosphorylation, glycosylation,
sumoylation, ubiquination
DNA and protein are packed into discrete units
called nucleosomes
Changes in Chromosome Structure
During the Cell Cycle
Nucleosomes consist of DNA wrapped
around histones

DNA of length 105 m fits into nucleus of

diameter 510 m
Partial unfolding reveals beads on a string
Beads are ~146 bp of DNA wrapped around 8 histones
(the core)
Forms left-handed solenoid
String is linker DNA of ~54 bp bound to histone H1
Amino-terminal tails of histones stick out, form sites for
covalent modification, and form important contacts
between nucleosomes
Nucleosomes
Nucleosomal DNA is underwound

Wrapping DNA around the histone core requires

removal of one helical turn
The underwinding occurs without a strand break, so a
compensatory (+) supercoil forms
This (+) supercoil is relaxed by a topoisomerase, leaves
DNA with net negative supercoil
DNA Wrapped Around a Histone Core
Front and Side Views of
Histone Amino-Terminal
Tails
How Amino-Terminal Tails Interact
between Nucleosomes
 Nucleosomes then assemble into
higher-order structures
Nucleosome formation compacts DNA seven-foldbut overall
compaction is >10K-fold!
Next level of structure: 30 nm fiber
Occurs in regions where sequence-specific, non-histone proteins bind
The 30 nm Fiber
Higher-level structures depend on
chromosomal scaffold
Higher-order structures are not well understood
May vary between chromosomes, regions, even
moment of time
Appears to involve a loop of DNA (maybe with
related genes) associating with a scaffold of
proteins
Includes Topo II and SMC proteins
SMC = Structural Maintenance of Chromosomes
Compaction of DNA in a Eukaryotic
Chromosome
 Condensed chromosome structure is
maintained by SMC proteins

SMC proteins have five domains

N and C domains form ATP-binding site
 Structure of SMC Proteins
Two major types
Cohesins help link sister chromatids
Condensins help chromosomes to condense; create positive supercoils
Roles of Cohesins and Condensins in
the Cell Cycle
 Bacterial DNA is organized into
nucleoids

Can occupy much of cell volume

DNA attaches to plasma membrane
Scaffold-like structure organizes the circular DNA
into ~500 looped domains
DNA binds to proteins transiently
Example: Protein HU
E. coli Nucleoids
Looped Domains of the E. coli
Chromosome
 Summary
In this chapter, we learned:
Genetic information of the cell is encoded in the nucleotide
sequence of one or several DNA molecules
The protein-coding regions represent only a small fraction of the
total DNA
Telomeres and centromeres regulate cell division
Bacterial DNA is usually supercoiled for efficient packing
Eukaryotic DNA is wound around positively charged histones
Higher-order organization of chromatin likely involves coils upon
coils upon coils