By Dr. Chandrama P. Upadhyaya Chapter 4 Chromatin and chromosomes By Dr. Chandrama P. Upadhyaya

Chromosomes vs. Chromatin Unwound DNA Active throughout Interphase DNA is being used for macromolecule synthesis Chromosomes Tightly packaged DNA Very active during cell division DNA is not being used for macromolecule synthesis

Chromosomes & Genomes Chromosomes Genome complexes of DNA and proteins – chromatin Viral – linear, circular; DNA or RNA Bacteria – single, circular Eukaryotes – multiple, linear Genome The genetic material that an organism possesses Nuclear genome Mitochondrial & chloroplasts genome

Chromosome in viruses Genome is Infectious particles containing nucleic acid surrounded by a protein capsid. Rely on host cell for replication, transcription, translation Exhibit a limited host range Genomes vary from a few thousand to a hundred thousand nucleotides

Some Virus Structures Phage Capsid protein TMV

Bacterial Chromosomes In a region called the nucleoid DNA in direct contact with cytoplasm

Bacterial Chromosomes Chromosomal DNA is compacted ~ 1000 fold to fit within cell

Bacterial Chromosomes Size Escherichia coli ~ 4.6 million bp Haemophilus influenzae ~ 1.8 million bp Composition E coli ~6000 genes Genes encoding proteins for related functions arranged in operons Intergenic regions nontranscribed DNA Single origin of replication (Ori)

Prokaryotic Gene (Operon) Structure Stop Codon TAA, TAG, TGA Regulatory Elements Cistron 1 Coding Sequence= ORF +1 ATG Cistron 2 Promoter & Operator DNA Terminator sequence Regulatory and Coding Sequence Unit = Operon Protein A Protein B Structural or Coding Sequences Regulatory Sequences

Eukaryotic Chromosomes Eukaryotic species contain one or more sets of chromosomes (ploidy level) Each set is composed of several linear chromosomes DNA amount in eukaryotic species is greater than that in bacteria Chromosomes in eukaryotes are located in the nucleus To fit in there, they must be highly compacted This is accomplished by the binding of many proteins The DNA-protein complex is termed chromatin 10-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic genomes vary substantially in size variation not related to complexity of the species i.e - a two fold difference in genome size between two salamander species Size difference due to accumulation of repetitive DNA sequences

Variations in DNA Content Figure 10.10

Eukaryotic Chromosome Organization Eukaryotic chromosomes are long, linear DNA molecule Three types of DNA sequences are required for chromosome replication and segregation Origins of replication (multiple) Centromeres (1) Telomeres (2)

Centromere Kinetochore proteins Origin of replication Telomere Genes Repetitive sequences

Chromosome Organization Genes located between centromere & telomeres hundreds to thousands of genes lower eukaryotes (i.e. yeast) Genes are relatively small Very few introns higher eukaryotes (i.e. mammals) Genes are long Have many introns Non-gene sequences Repetitive DNA Telomere Centromere Satellite

Eukaryotic Gene Structure Start Codon ATG Stop Codon TAA, TAG, TGA Cis-Regulatory Elements Exon1 Exon2 Exon3 Promoter/ Enhancer

Repetitive Sequences Sequence complexity refers to the number of times a particular base sequence appears in the genome 2 main types of sequences Moderately repetitive Highly repetitive (low complexity)

Repetitive Sequences Unique or non-repetitive sequences Found once or a few times in the genome Includes structural genes as well as intergenic areas Moderately repetitive Found a few hundred to a few thousand times Includes Genes for rRNA and histones Origins of replication Transposable elements 10-28

Repetitive Sequences Highly repetitive Found tens of thousands to millions of times Each copy is relatively short (a few nucleotides to several hundred in length) Some sequences are interspersed throughout the genome Example: Alu family in humans Other sequences are clustered together in tandem arrays Example: centromeric satellite & telomeric regions

Eukaryotic Chromatin Compaction Stretched end to end, a single set of human chromosomes will be over 1 meter long nucleus is only 2 to 4 mm in diameter The compaction of linear DNA in eukaryotic chromosomes involves interactions between DNA and various proteins Proteins bound to DNA are subject to change during the life of the cell These changes affect the degree of chromatin compaction

Nucleosomes Histone proteins basic (+ charged lysine & arginine) amino acids that bind DNA backbone Four core histones in nucleosome Two of each of H2A, H2B, H3 & H4 Fifth histone, H1 is the linker histone Figure 10.14

Nucleosomes Figure 10.14

Beads on a String Shortens DNA length ~ seven-fold Overall structure of connected nucleosomes resembles “beads on a string” Shortens DNA length ~ seven-fold

Nucleosomes Join to Form 30 nm Fiber Nucleosomes associate to form more compact structure - the 30 nm fiber Histone H1 plays a role in this compaction 10-54

Further Compaction of the Chromosome The two events we have discussed so far have shortened the DNA about 50-fold A third level of compaction involves interaction between the 30 nm fiber and the nuclear matrix 10-57 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Nuclear Matrix Association Nuclear matrix composed of two parts Nuclear lamina Internal matrix proteins 10 nm fiber and associated proteins Figure 10.18

DNA Loops on Nuclear Matrix The third mechanism of DNA compaction involves the formation of radial loop domains Figure 10.18 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

Further Compaction of the Chromosome The attachment of radial loops to the nuclear matrix is important in two ways 1. It plays a role in gene regulation Discussed in Chapter 15 2. It serves to organize the chromosomes within the nucleus Each chromosome in the nucleus is located in a discrete and nonoverlapping chromosome territory Refer to Figure 10.19 10-60 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Heterochromatin vs Euchromatin Compaction level of interphase chromosomes is not uniform Euchromatin Less condensed regions of chromosomes Transcriptionally active Regions where 30 nm fiber forms radial loop domains Heterochromatin Tightly compacted regions of chromosomes Transcriptionally inactive (in general) Radial loop domains compacted even further

Types of Heterochromatin Figure 10.20 Constitutive heterochromatin Always heterochromatic Permanently inactive with regard to transcription Facultative heterochromatin Regions that can interconvert between euchromatin and heterochromatin Example: Barr body

Figure 10.21

10-64 Figure 10.21 Compaction level in euchromatin During interphase most chromosomal regions are euchromatic Compaction level in heterochromatin Figure 10.21 10-64

Metaphase Chromosomes Condensed chromosomes are referred to as metaphase chromosomes During prophase, the compaction level increases By the end of prophase, sister chromatids are entirely heterochromatic These highly condensed metaphase chromosomes undergo little gene transcription In metaphase chromosomes, the radial loops are compacted and anchored to the nuclear matrix scaffold

Chromosome Condensation The number of loops has not changed However, the diameter of each loop is smaller During interphase, condensin is in the cytoplasm Condensin binds to chromosomes and compacts the radial loops Condensin travels into the nucleus The condensation of a metaphase chromosome by condensin Figure 10.23

Chromosomes During Mitosis Cohesins along chromosome arms are released Cohesin at centromer is degraded Cohesin remains at centromere The alignment of sister chromatids via cohesin

Chromosome condensation is caused by condensins SMC proteins are ATPases that include the condensins and the cohesins. A heterodimer of SMC proteins associates with other subunits. Cohesins are responsible for holding sister chromatids together. Condensins cause chromatin to be more tightly coiled by introducing positive supercoils into DNA.

Condensins could compact DNA. Condensins are responsible for condensing chromosomes at mitosis. Chromosome-specific condensins are responsible for condensing inactive X chromosomes in C. elegans. Condensins could compact DNA.