CHAPTER 24 Genes and Chromosomes

Organization of information in chromosomes DNA supercoiling Structure of the chromosome

Key topics:

Management and Expression of Genetic Information

Previous chapters dealt with metabolic pathways, in which the chemical

structures of small molecules were modified by enzymes

The following chapters deal with information pathways, in which genetic

information stored as the nucleotide sequence is maintained and expressed

The Central Dogma of Molecular Biology

The discovery of double-helical structure of DNA in 1953 laid a foundation to thinking of biomolecules as carriers of information

It was well understood by 1950 that proteins play roles of catalysts but their role in information transfer was unclear

Francis Crick proposed in 1956 that “Once information has got into a protein it can’t get out again”

The Central Dogma was proposed by Francis Crick at the time when there was little evidence to support it, hence the “dogma”

How does genes function?

Central Dogma: DNA to RNA to Protein.

Genes and Chromosomes

What is gene? One gene-one enzyme. One gene-one protein (polypeptide). Genes are segments of DNA that code for polypeptides an

d RNAs. What is chromosome?

Chromosome consists of one covalently connected DNA molecule and associated 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 calle

d the chromatin

DNA is a Very Large Macromolecule

The linear dimensions of DNA are much bigger than the virions or cells that contain them Bacteriophages T2 and T4 are about 0.2 m long and 0.1

m wide Fully extended T4 DNA double helix is about 60 m long

DNA in the virion or cell is organized into compact forms, typically via coiling and association with proteins

The Size and Sequence of DNA Molecules in Bacteria and their viruses

Bacteria(E. coli) 4,639,221 1.7 mm 0.002 mm

T2 phage

The sizes of E. coli cell and its DNA

DNA from a lysed E. coli cell

DNA content and C-value paradox

DNA, Chromosomes, Genes, and Complexity

Note that despite the trends in the previous table, neither the total length of DNA, nor the number of chromosomes correlates strongly with the perceived complexity of the organisms

Amphibians have much more DNA than humans Dogs and coyotes have 78 chromosomes in the diploid cell Plants have more genes than humans

The correlation between complexity and genome size is poor because most of eukaryotic DNA is non-coding

Recent experimental work by Craig Venter suggests that a minimal living organisms could get by with less than 400 genes

Eukaryotic genomes have several sequence components Nonrepetitive DNA: the complexity of the slow component cor

responds with its physical size, i.e., unique sequences. Moderately repetitive DNA:.component with a Cot1/2 of 10-2 a

nd that of nonrepetitive DNA. Contains families of sequences that are not exactly the same, but are related. The complexity is made up of a variety of individual sequences, each much shorter, whose total length together comes to the putative complexity. Usually dispersed throughout the genome.

Highly repetitive DNA: component which reassociates before a Cot1/2 of 10-2. Usually forms discrete clusters.

Types of sequences in the human genome

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 all 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

Many eukaryotic genes contain intervening sequences (introns)

Some Bacterial Genomes Also Contain Introns

It was thought until 1993 that introns are exclusive feature of eukaryotic genes

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 for catalytic RNA molecules that have ability to insert and reverse transcribe themselves into the genomic DNA

Transposons DNA sequence is not completely static Some sequences, called transposons, can move aro

und within the genome of a single cell The ends of transposons contain terminal repeats th

at hybridize with the complementary regions of the target DNA during insertion

To be covered in Ch. 25.

Eukaryotic Chromosomes

Important Structural Elements of the Eukaryotic Chromosome

Telomeres cap the ends of linear chromosomes and are needed for successful cell division

Centromere functions in cell division; that’s where the two daughter chromosomes are held together during mitosis (i.e. after DNA replication but before cell division)

Centromere: Mitotic segregation of chromosomes. Simple-sequence DNA is located at centromere in higher eukaryotes.

Telomere: At ends of chromosomes. (TTAGGG)n in human

YAC: Yeast artificial chromosome requires only yeast centromere, telomere and replication origin (eg., ARS).

Telomeres and Cellular Aging

In many tissues, telomeres are shortened after each round of replication (end-replication problem of linear DNA); the cellular DNA ages

Normal human cells divide about 52 times before losing ability to divide again (Hayflick limit)

How is DNA packed in the chromosomes

DNA Supercoiling. Proteins assisted packaging (nucleosomes)

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 sequen

ce

There are several levels of organization, one of whi

ch is the supercoiling of the double-stranded DNA h

elix

Supercoils

Supercoiling of DNA can only occur in closed-circular DNA or linear DNA where the ends are fixed.

Underwinding produces negative supercoils, wheres overwinding produces positive supercoils.

Negative and positive supercoils .

Topoisomerases catalyze changes in the linking number of DNA.

Supercoiling induced by separating the strands of duplex DNA (eg., during DNA replication)

The Effects of Replication and Transcription on DNA Supercoiling

Relaxed and supercoiled plasmid DNAs

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 eas

ier Linear DNA is underwound with the help of protei

ns to prevent strands from rotating

Topology of cccDNA is defined by: Lk = Tw + Wr, where Lk is the linking number, Tw is twist and Wr is writhe.

Intertwining of the two strands Nodes = ss crossing on 2D projection.

Right-handed crossing = +1/2

Left-handed crossing = -1/2

Lk = number of times one strand winds around the other on 2D projection.One linking number = 2 nodes.

Linking number (Lk) describes supercoiling• In circular DNA, changing the helical turns requires bre

aking 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.

Superhelical Density,

Negative supercoils facilitate separation of DNA strands (may facilitate transcription)

Promotion of cruciform structures by DNA underwinding

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

Mechanism of Type I topoisomerase action

Proposed mechanism of Type II topoisomerase action

Topoisomerases are Targets for Antibiotics and Anti-cancer Drugs

Bacterial topoisomeraseinhibitors

Type I topoisomeraseinhibitors

Human Type II topoisomerase inhibitors

DNA damages are produced by topoisomerase inhibitors

Most topoisomerase inhibitors act by blocking the last step of the topoisomerase reaction, the resealing of the DNA strand breaks. Therefore, these inhibitors will produce single-strand or double-strand DNA breaks in the DNA.

Plectonemic supercoiling

DNA Compaction Requires Solenoidal Supercoiling, not plectonemic supercoiling.

Changes in Chromosome Structure During the Cell Cycle

Changes in chromosome structure during the cell cycle

Protein-assisted Packaging of DNA

Nucleosomes are the fundamental organizational units of eukaryotic chromatin

Each nucleosome has a histone core wrapped by DNA (146 bps) in a left-handed solenoidal supercoil about 1.8 times. The linker DNA is about 54 bps in length.

DNA wrapped around a nucleosome core

DNA Wrapped Around a Histone Core

Front and Side Views of Histone Amino-Terminal Tails

Histones are small, basic protein. The histone core in nucleosomes contains two copies each of H2A, H2B, H3 and H4. Histone H1 binds to linker DNA.

Histone binding depends on DNA sequence Histone binding is not random Occurs more often at A-T–rich regions Staggering AA, AT or TT at 10 bp intervals (phased with pitch of helix) narrows the minor groove, bends the DNA

facilitates its binding around the histone core

Effect of DNA Sequence on Nucleosome Binding

Chromatin assembly

Nucleosomes are packed into successively higher-order structures

The 30 nm fiber, a higher-order organization of nucleosomes.

The 30 nm Fiber

A partially unraveled human chromosome, revealing numerous loops of DNA attached to scaffold.

Loops of DNA Attached to a Chromosomal Scaffold

Higher order of folding is not yet understood. Certain regions of DNA are associated with a nuclear scaffold. The scaffold associated regions are separated by loops of DNA with 20 to 100 kb long.

Compaction of DNA in a Eukaryotic Chromosome

Model of DNA compaction in eukaryotic chromosomes

Condensed chromosome are maintained by SMC proteins

SMC Proteins

Structure of SMC Proteins

Model for the effect of condensins on DNA supercoiling

Possible Role of Condensins

Bacterial DNA is organized into nucleoids

Can occupy much of cell volume DNA attaches to plasma membrane Scaffold-like structure organizes the circular DNA i

nto ~500 looped domains DNA binds to proteins transiently

Example: Protein HU

Looped Domains of the E. coli Chromosome