Chromosomestructure

Genetics: Analysis and Genetics: Analysis and PrinciplesPrinciples

CHAPTER 10CHAPTER 10

CHROMOSOME ORGANIZATION CHROMOSOME ORGANIZATION AND MOLECULAR STRUCTUREAND MOLECULAR STRUCTURE

By Robert J. BrookerBy Robert J. Brooker

In which form does DNA and In which form does DNA and RNA occur in a cell?RNA occur in a cell?

Never naked!Never naked!

Always associated with proteinsAlways associated with proteins From small virus genome to big genome of From small virus genome to big genome of

a complex organisme. a complex organisme. Proteins associated with DNA play a Proteins associated with DNA play a

significant role in regulation of gene significant role in regulation of gene expression/repression.expression/repression.

Viruses are small infectious particles containing nucleic acid Viruses are small infectious particles containing nucleic acid surrounded by a capsid of proteinssurrounded by a capsid of proteins

For replication, viruses rely on their For replication, viruses rely on their host cellshost cells ie., the cells they infectie., the cells they infect

Most viruses exhibit a limited Most viruses exhibit a limited host rangehost range They typically infect only specific types of cells of one host speciesThey typically infect only specific types of cells of one host species

VIRAL GENOMESVIRAL GENOMES

Animal virussesAnimal virussesLipid bilayer

Picked up when virus leaves host cell

A A viral genomeviral genome is the genetic material of the virus is the genetic material of the virus Also termed the Also termed the viral chromosomeviral chromosome

The genome can beThe genome can be DNA or RNADNA or RNA Single-stranded or double-strandedSingle-stranded or double-stranded Circular or linearCircular or linear

Viral genomes vary in size from a Viral genomes vary in size from a few thousandfew thousand to more to more than a than a hundred thousandhundred thousand nucleotides nucleotides

Viral GenomesViral Genomes

Figure 10.1 General structure of viruses

Bacteriophages may also contain a sheath, base plate and tail fibers

Lipid bilayer

Picked up when virus leaves host cell

During an infection process, mature viral particles During an infection process, mature viral particles need to be assembledneed to be assembled

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Viruses with a simple structure may self-assemble Genetic material

and capsid proteins spontaneously bind to each other

Example: Tobacco mosaic virus

Figure 10.2 Capsid composed of 2,130 identical protein subunits

Complex viruses, such as T2 bacteriophages, Complex viruses, such as T2 bacteriophages, undergo a process called undergo a process called directed assemblydirected assembly Virus assembly requires proteins that are not part of the Virus assembly requires proteins that are not part of the

mature virus itself; Helper proteins / “chaperone mature virus itself; Helper proteins / “chaperone proteins”proteins”

The noncapsid proteins usually have two main The noncapsid proteins usually have two main functionsfunctions 1. Carry out the assembly process1. Carry out the assembly process

• Scaffolding proteins that are not part of the mature virusScaffolding proteins that are not part of the mature virus

2. Act as proteases that cleave viral capsid proteins2. Act as proteases that cleave viral capsid proteins• This yields smaller capsid proteins that assemble correctlyThis yields smaller capsid proteins that assemble correctly

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The bacterial chromosome is found in a region called the The bacterial chromosome is found in a region called the nucleoidnucleoid

The nucleoid is not membrane-boundedThe nucleoid is not membrane-boundedSo the DNA is in direct contact with the cytoplasm So the DNA is in direct contact with the cytoplasm

BACTERIAL CHROMOSOMESBACTERIAL CHROMOSOMES

DNA supercoilingDNA supercoiling is a second important way to is a second important way to compact the bacterial chromosomecompact the bacterial chromosome

Figure 10.6

Figure 10.7 provides a schematic illustration of Figure 10.7 provides a schematic illustration of DNA supercoilingDNA supercoiling

Supercoiling within loops creates a more compact DNA

Terms and concepts to know:

CompactionCoilingSupercoilingGyrasetopoisomerase

Figure 10.7Plates preventing DNA

ends from rotating freely

Fewer turnsMore turns

Both overwinding and underwinding can

induce supercoiling

These three DNA conformations are topoisomers of each other

These two DNA conformations do not occur in living cells

Increasing of ‘coiling’ of the double helix in eukaryotes also occur:

- DNA replication

- transcription, RNA synthesis from a double stranded

DNA

Figure 10.8

It makes transcription and translation easier, because the chains melt easier.

negative supercoilingnegative supercoiling

Does not only lead to a more compacted nucleoid

1. 1. DNA gyraseDNA gyrase (also termed (also termed DNA topoisomerase IIDNA topoisomerase II))• Introduces negative supercoils using energy from Introduces negative supercoils using energy from

ATPATP• It can also relax positive supercoils when they occurIt can also relax positive supercoils when they occur

2. 2. DNA topoisomerase I DNA topoisomerase I • Relaxes negative supercoilsRelaxes negative supercoils

Supercoiling is regulated by two Supercoiling is regulated by two types of enzymes:types of enzymes:

Eukaryotic species contain one or more sets of Eukaryotic species contain one or more sets of chromosomeschromosomes

Each set is composed of several different linear chromosomesEach set is composed of several different linear chromosomes

The total amount of DNA in eukaryotic species is The total amount of DNA in eukaryotic species is typically greater than that in bacterial cellstypically greater than that in bacterial cells

Chromosomes in eukaryotes are located in the Chromosomes in eukaryotes are located in the nucleusnucleus To fit in there, they must be highly compactedTo fit in there, they must be highly compacted

• This is accomplished by the binding of many proteinsThis is accomplished by the binding of many proteins• The DNA-protein complex is termed The DNA-protein complex is termed chromatin chromatin

EUKARYOTIC CHROMOSOMESEUKARYOTIC CHROMOSOMES

Has a genome that is more than twice as large as that

of

Genome size is very various, especially Genome size is very various, especially because of the presence of Repetitive DNAbecause of the presence of Repetitive DNA

A eukaryotic chromosome contains a long, linear DNA A eukaryotic chromosome contains a long, linear DNA moleculemolecule

Refer to Figure 10.11Refer to Figure 10.11

Three types of DNA sequences are required for Three types of DNA sequences are required for chromosomal replication and segregationchromosomal replication and segregation

Origins of replicationOrigins of replication CentromeresCentromeres TelomeresTelomeres

Organization of Eukaryotic Organization of Eukaryotic ChromosomesChromosomes

Figure 10.11

Prevent chromosome sticky ends and shortening

Required for proper segregation during mitosis and meiosis

Eukaryotic chromosomes contain many origins of

replication approximately 100,000 bp apart

Genes are located between the Genes are located between the centromericcentromeric and and telomerictelomeric regions regions along the entirealong the entire chromosome chromosome

A single chromosome usually has a A single chromosome usually has a few hundredfew hundred to to several several thousandthousand genes genes

In In lower eukaryoteslower eukaryotes (such as yeast) (such as yeast) Genes are Genes are relatively smallrelatively small

• They contain primarily the sequences encoding the polypeptidesThey contain primarily the sequences encoding the polypeptides• ie: Very ie: Very few intronsfew introns are present are present

In In higher eukaryoteshigher eukaryotes (such as mammals) (such as mammals) Genes are Genes are longlong

• They tend to have They tend to have many intronsmany introns• intron lengths from less than intron lengths from less than 100100 to more than to more than 10,00010,000 bp bp

General features of eukaryotic chromosomesGeneral features of eukaryotic chromosomes

Sequence complexitySequence complexity refers to the number of refers to the number of times a particular base sequence appears in times a particular base sequence appears in the genomethe genome

There are three main types of repetitive There are three main types of repetitive sequencessequences Unique or non-repetitiveUnique or non-repetitive Moderately repetitiveModerately repetitive Highly repetitiveHighly repetitive

Repetitive SequencesRepetitive Sequences

Unique or non-repetitive sequencesUnique or non-repetitive sequences Found once or a few times in the genomeFound once or a few times in the genome Includes structural genes as well as intergenic areasIncludes structural genes as well as intergenic areas

Moderately repetitiveModerately repetitive Found a few hundred to a few thousand times Found a few hundred to a few thousand times Includes Includes

• Genes for rRNA and histonesGenes for rRNA and histones• Origins of replicationOrigins of replication• Transposable elementsTransposable elements

Highly repetitiveHighly repetitive Found tens of thousands to millions of timesFound tens of thousands to millions of times Each copy is relatively short (a few nucleotides to several hundred in Each copy is relatively short (a few nucleotides to several hundred in

length)length)

Repetitive SequencesRepetitive Sequences

If If stretchedstretched end to end, a doploid set of human end to end, a doploid set of human chromosomes will be over chromosomes will be over 2 meter 2 meter long! long! Yet the cell’s nucleus is only Yet the cell’s nucleus is only 2 to 4 2 to 4 mm in diameter in diameter

• Therefore, the DNA must be tightly compacted to fit Therefore, the DNA must be tightly compacted to fit

The The compaction of linear DNAcompaction of linear DNA in eukaryotic in eukaryotic chromosomes involves interactions between DNA chromosomes involves interactions between DNA and various proteins and various proteins Proteins bound to DNA are subject to change during the Proteins bound to DNA are subject to change during the

life of the celllife of the cell• These changes affect the degree of chromatin compaction These changes affect the degree of chromatin compaction

Eukaryotic Chromatin Eukaryotic Chromatin CompactionCompaction

Eukaryotic Chromatin CompactionEukaryotic Chromatin Compaction

``````Samenstelling relatieve hoeveelheid

Histon eiwitten 115

Non-histon eiwitten 33

RNA 1

100DNA

Histone proteinsHistone proteins are basic are basic They contain many positively-charged amino acidsThey contain many positively-charged amino acids

• Lysine and arginineLysine and arginine

These bind with the phosphates along the DNA backboneThese bind with the phosphates along the DNA backbone Histone proteins have a globular domain and a flexible, Histone proteins have a globular domain and a flexible,

charged amino terminus or ‘tail’ charged amino terminus or ‘tail’

There are five types of histonesThere are five types of histones H2A, H2B, H3 and H4H2A, H2B, H3 and H4 are the are the core histonescore histones

• Two of each make up the octamerTwo of each make up the octamer

H1 is the linker histoneH1 is the linker histone• Binds to linker DNABinds to linker DNA• Also binds to nucleosomesAlso binds to nucleosomes

But not as tightly as are the core histonesBut not as tightly as are the core histones


Overall structure of connected nucleosomes resembles “beads on a Overall structure of connected nucleosomes resembles “beads on a string”string” This structure shortens the DNA length about seven-foldThis structure shortens the DNA length about seven-fold

Figure 10.13

Vary in length between 20 to 100 bp, depending on species and cell type

Diameter of the nucleosome

Figure 10.13

Play a role in the organization and compaction

of the chromosome

Figure 10.16

Regular, spiral configuration containing six nucleosomes

per turn

Irregular configuration where nucleosomes have little face-to-face

contact

The model of nucleosome structure was proposed The model of nucleosome structure was proposed in 1974 by Roger Kornbergin 1974 by Roger Kornberg

Kornberg based his proposal on various Kornberg based his proposal on various observations about chromatinobservations about chromatin Biochemical experimentsBiochemical experiments X-ray diffraction studiesX-ray diffraction studies Electron microscopy imagesElectron microscopy images


Experiment I: Nucleosome Experiment I: Nucleosome Structure RevealedStructure Revealed

Markus Noll decided to test Kornberg’s model Markus Noll decided to test Kornberg’s model

He did this via the following procedureHe did this via the following procedure Digest DNA with the enzyme DNase I Digest DNA with the enzyme DNase I Accurately measure the molecular weight of the Accurately measure the molecular weight of the

resulting DNA fragmentsresulting DNA fragments

The rationale is that the linker DNA is more accessible The rationale is that the linker DNA is more accessible than the “core DNA” to the DNase Ithan the “core DNA” to the DNase I

• Thus, the cuts made by DNase I should occur in the linker DNA Thus, the cuts made by DNase I should occur in the linker DNA


Experiment II: Nucleosome Experiment II: Nucleosome Structure RevealedStructure Revealed

The HypothesisThe Hypothesis

The experiment tests the beads-on-a-string model The experiment tests the beads-on-a-string model of chromatin structure of chromatin structure • It the model is correct, DNase I should cut in the linker It the model is correct, DNase I should cut in the linker

regionregion Thereby producing DNA pieces that are about 200 bp longThereby producing DNA pieces that are about 200 bp long


Figure 10.14

The Data


30 unitsml-1

Interpreting the Data


30 unitsml-1

All chromosomal DNA digested into fragments that are ~ 200 bp in length

These longer pieces were all in multiples of 200 bp

At low concentrations, DNase I did not cut at all

the linker DNA

This fragment contains two nucleosomes

And this, three

Figure 10.15aFigure 10.15a

‘BEADS ON A STRING’

Figure 10.15bFigure 10.15b

‘BEADS ON A STRING’+ linker histone H1


Nucleosomes associate with each other to form a more compact structure termed the 30 nm fiber

Histone H1 plays a role in this compaction At moderate salt concentrations, H1 is removed

The result is the classic beads-on-a-string morphology At low salt concentrations, H1 remains bound

Beads associate together into a more compact morphology

Nucleosomes Join to Form Nucleosomes Join to Form a 30 nm Fibera 30 nm Fiber

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Figure 10.17

The third mechanism of DNA compaction involves the formation of radial loop domains

Matrix-attachment regions

Scaffold-attachment regions (SARs)

or

MARs are anchored to the nuclear

matrix, thus creating radial loops

25,000 to 200,000 bp

The levels of compaction leading to a metaphase chromosome

The levels of compaction leading to a metaphase chromosome

Figure 10.21

Figure 10.21

Compaction level in euchromatin

Compaction level in heterochromatin

During interphase most chromosomal

regions are euchromatic

The compaction level of interphase chromosomes The compaction level of interphase chromosomes is not completely uniformis not completely uniform EuchromatinEuchromatin

• Less condensed regions of chromosomesLess condensed regions of chromosomes• Transcriptionally activeTranscriptionally active• Regions where 30 nm fiber forms radial loop domainsRegions where 30 nm fiber forms radial loop domains

HeterochromatinHeterochromatin• Tightly compacted regions of chromosomesTightly compacted regions of chromosomes• Transcriptionally inactive (in general)Transcriptionally inactive (in general)• Radial loop domains compacted even furtherRadial loop domains compacted even further

Heterochromatin Heterochromatin vsvs EuchromatinEuchromatin

There are two types of heterochromatinThere are two types of heterochromatin Constitutive heterochromatinConstitutive heterochromatin

• Regions that are always heterochromaticRegions that are always heterochromatic• Permanently inactive with regard to transcriptionPermanently inactive with regard to transcription

Facultative heterochromatinFacultative heterochromatin• Regions that can interconvert between euchromatin and Regions that can interconvert between euchromatin and

heterochromatinheterochromatin• Example: Barr bodyExample: Barr body

Figure 10.19

The compaction level of even euchromatin is too The compaction level of even euchromatin is too high for transcription factors and RNA polymerase high for transcription factors and RNA polymerase to easily access and transcribe genesto easily access and transcribe genes Chromatin remodelingChromatin remodeling changes chromatin structure changes chromatin structure

• regulates ability of transcription factors to access genesregulates ability of transcription factors to access genes

Histone core protein tails are modifiedHistone core protein tails are modified• over 50 different enzymes identified which modify tailsover 50 different enzymes identified which modify tails• modifications include acetylation, methylation and modifications include acetylation, methylation and

phosphorylation-all covalent changesphosphorylation-all covalent changes• refer to figure 10.20refer to figure 10.20

Histone code hypothesisHistone code hypothesis is that the pattern of is that the pattern of modification is a code specifying alterationsmodification is a code specifying alterations

Histone Code Controls Histone Code Controls CompactionCompaction

Figure 10.20

The histon-codeThe histon-code

1. Lysines may be acetylated1. Lysines may be acetylated

2. Serines may be phoshorylated2. Serines may be phoshorylated

3. arginines may be methylated3. arginines may be methylated

As cells enter M phase, the level of compaction As cells enter M phase, the level of compaction changes dramaticallychanges dramatically By the end of prophase, sister chromatids are entirely By the end of prophase, sister chromatids are entirely

heterochromaticheterochromatic Two parallel chromatids have an overall diameter of Two parallel chromatids have an overall diameter of

1,400 nm 1,400 nm

These highly condensed metaphase chromosomes These highly condensed metaphase chromosomes undergo little gene transcriptionundergo little gene transcription

Metaphase ChromosomesMetaphase Chromosomes

Eukaryotic Chromosome

In metaphase chromosomes the radial loops are In metaphase chromosomes the radial loops are highly compacted and stay anchored to a highly compacted and stay anchored to a scaffoldscaffold The scaffold is formed from the nuclear matrixThe scaffold is formed from the nuclear matrix

Histones are needed for the compaction of radial Histones are needed for the compaction of radial loopsloops




Figure 10.22 (The scaffold)

Two multiprotein complexes help to form and Two multiprotein complexes help to form and organize metaphase chromosomesorganize metaphase chromosomes

CondensinCondensin• Plays a critical role in chromosome condensationPlays a critical role in chromosome condensation

CohesinCohesin• Plays a critical role in sister chromatid alignmentPlays a critical role in sister chromatid alignment

Both contain a category of proteins called Both contain a category of proteins called SMC SMC proteinsproteins Acronym = Acronym = SStructural tructural mmaintenance of aintenance of cchromosomeshromosomes SMC proteins use energy from ATP and catalyze SMC proteins use energy from ATP and catalyze

changes in chromosome structurechanges in chromosome structure

The alignment of sister chromatids via cohesin

Cohesins along chromosome arms

are released

The condensation of a metaphase chromosome by condensin

Model !

The number of loops has not changedHowever, the diameter of each loop is smaller

Condesin travels into the nucleus

Condesin binds to chromosomes and

compacts the radial loops

During interphase, condensin is in the cytoplasm

‘‘Chromosome territories’Chromosome territories’chromosomen nemen een bepaalde ruimte chromosomen nemen een bepaalde ruimte

in in de nucleus: Geen spaghettiin in de nucleus: Geen spaghetti

Chromosome of a chicken

Nucleus where eachchromosome is colored differently

Viral Genomes - Viral genomes are relatively small and are composed of DNA or RNA - Viral genomes are packaged into the capsid in an assembly process

Bacterial Chromosomes - Bacterial chromosomes contain a few thousand gene sequences that are interspersed with other functionally important sequences - The formation of chromosomal loops helps make the bacterial chromosome more compact- DNA supercoiling further compacts the bacterial chromosome- Chromosome function is influenced by DNA supercoiling

Eukaryotic Chromosomes - The sizes of eukaryotic genomes vary substantially- Eukaryotic chromosomes have many functionally important sequences including genes, origins of replication, centromeres, and telomeres - The genomes of eukaryotes contains sequences that are unique, moderately repetitive, or highly repetitive - Sequence complexity can be evaluated in a renaturation experiment- Eukaryotic chromatin must be compacted to fit within the cell- Linear DNA wraps around histone proteins to form nucleosomes, the repeating structural unit of chromatin- The repeating nucleosome structure is revealed by digestion of the linker region- Nucleosomes become closely associated to form a 30 nm fiber- Chromosomes are further compacted by anchoring the 30 nm fiber into radial loop domains along the nuclear matrix- The histone code controls chromatin compaction- Condensin and cohesin promote the formation of metaphase chromosomes

Summary / study outline

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Chromosomestructure