Chromosome Structure and DNA Organization in Eukaryotes and Prokaryotes
DNA Organization in Chromosomes: DNA to Chromatin to Chromosomes
Viral and Bacterial Chromosomes: Much simpler than those of eukaryotes, single DNA molecule.
Prokaryotic Chromosomes: No associated proteins, less genetic information.
Virus Chromosome: Single or double-stranded DNA, can be a covalently closed circle (X174 & Polyoma), can be linear (T-even 2x).
- Bacteriophage λ is a linear double-stranded DNA molecule before infection, closes into a ring after infection of the host. Long DNA molecule put into a small volume.
Virus genetic material is inert until released into the host cell.
Bacterial Chromosomes: Double-stranded, compacted into a nucleotide. DNA associated with several DNA-binding proteins. Examples: HU and H1 proteins, small but abundant, high percentage of charged amino acids that can bond ionically to negative charges of the phosphate group in DNA. Similar to histones but do not compact DNA. Not functionally inert, can be replicated and transcribed.
Supercoiled DNA is characteristic of closed-circular molecules. It contorts in a certain way to retain normal base pairing. Caused by the energetic forces that stabilize the double helix to resist underwinding. This shape causes tighter packing and increases sedimentation velocity. Facilitates chromosome condensation in the nucleotide region.
Transition to Supercoil:
- Start with a regular linear double-stranded helix, (Watson and Crick right-handed), 20 complete turns, linking number (L) = 20, L = #bp/bp per complete turn.
- Closed circle, L = 20 (still), energetically relaxed.
- Underwound circle, circle cut, underwound two full turns, resealed, L = 18, energetically strained, will change form to relieve strain (will form supercoils).
- Supercoil, formed in the direction opposite of underwinding, 2 negative supercoils will form, negative since supercoils are left-handed on right-handed DNA, re-establishes the total number of turns, enhanced physical stability.
Virus SV40 example: 5200 bp, energetically relaxed at 10.4 bp per complete turn, L = 5200/10.4 = 500, analysis shows SV40 DNA underwound by 25 turns, L = 475.
Topoisomers: Two identical molecules that differ in linking number.
How do you convert from one topoisomer to another when no free ends in closed circle DNA?
Topoisomerases: Enzymes that cut one or both strands, wind or unwind, and reseal ends. Type I cleave one strand, type II cleave two strands.
In E. coli, topoisomerase I reduces the number of negative supercoils. Topoisomerase II introduces negative supercoils, binds to DNA, cleaves both strands, passes them through the loop it created, phosphodiester bonds reformed, L decreases, one or more supercoils form. Other functions: separate (decatenate) the DNA of sister chromatids following replication.
Supercoils are also found in eukaryotes. DNA is not usually circular. They occur in areas of DNA embedded in a lattice of protein associated with chromatin fibers, creating anchored ends. Introduce topoisomerases and you get supercoils.
In prokaryotes and eukaryotes, DNA replication and transcription create supercoils downstream as the helix unwinds and becomes accessible to the appropriate enzyme.
Specialized Chromosomes Reveal Variation in DNA Organization
Not found in most eukaryotic cells.
Polytene Chromosomes: Giant, found in various tissues of fly larvae, 200-600 µm long. Linear series of alternating bands (chromomeres) and interbands, pattern distinctive for each chromosome of a given species. Paired homologs, which is unusual since in somatic cells and not chromatin. Large size and distinctive appearance because of a large number of identical DNA strands. DNA undergoes many rounds of replication without strand separation, 1000-5000 DNA strands that remain in precise parallel alignment. Strands present in bands undergo localized uncoiling. Uncoiling event results in a puff, high-level gene activity (transcription that makes RNA).
Lampbrush Chromosomes: Characteristic of most vertebrate oocytes, and spermatocytes of some insects, meiotic chromosomes, homologs paired together by chiasmata, extended to 500-800 µm (during the first prophase), revert to 12-20 µm later in meiosis. Have chromomeres (large number of condensed areas), each chromomere has lateral loops jutting out. Much more DNA in a single loop than is needed for a single gene. Each loop is one double helix, each axis is two double helices. Loops are active in the synthesis of RNA. Like the puff, the loops are DNA reeled out from the central chromomere axis during transcription.
Eukaryotic DNA is Organized into Chromatin
Viewed during mitosis, interphase, components of chromosomes uncoil and decondense into chromatin, dispersed throughout the nucleus. As the cycle continues, it condenses into visible chromosomes (1/10000 of length change).
Chromatin Structure and Nucleosomes
Chromatin fibers: DNA and protein, ‘beads on a string’. Eukaryotic chromatin has a lot of associated proteins with chromosomal DNA: positively charged histones, less positively charged nonhistone proteins. Histones: Large amounts of lysine and arginine (+), can bond to (-) phosphate groups of nucleotides. DNA length reduced to 1/3 by wrapping around. Nucleosomes: “the beads”, octamer, made from tetramer (H2A)2 x (H2B)2, with tetramer (H3)2 x (H4)2, in association with 200 bp of DNA. Nucleosome core particle: 147 base pairs, the rest is linker DNA associated with the fifth histone, H1.
Packing Levels
- Chromatin fiber with nucleosomes
- Solenoid
- Looped domains
- Coiled chromatin fibers
Chromatin Remodeling
“In the chromatin fiber, complex with histones into nucleosomes, which may be further folded into several more levels of compaction, the DNA is inaccessible to interaction with important nonhistone proteins.” Chromatin remodeling is when chromatin’s structure is induced to change to allow access for protein-DNA interactions, and mechanisms for reversing the process during inactivity.
Histone tails: Unstructured, not packed into the folded histone domains of the nucleosome, protrude out, target for chemical modification.
Acetylation: Histone modification, by action of the enzyme histone acetyltransferase (HAT). Addition of an acetyl group to the (+) amino group of lysine. Neutralizes the positive charge, linked to gene activation. High levels of acetylation open up/remodel chromatin fiber, increase in areas of active genes, decrease in inactive regions.
Methylation: Histone modification, from enzyme methyltransferases, methyl groups added to arginine and lysine, correlated to gene activity. **When methylation happens of the nitrogenous base cytosine in polynucleotide chains of DNA, making 5-methyl cytosine is negatively correlated with gene activity, making CpG islands.
Phosphorylation: From enzyme kinases, phosphate groups added to hydroxyl groups of serine and histidine, adds a negative charge to the protein, happens to H3 for chromatin unfolding and condensation during and after DNA replication.
^^All are reversible with specific enzymes.
Epigenetics: The study of modifications of an organism’s genetic and phenotypic expression that are not attributable to alteration of the DNA sequence making up a gene.
Heterochromatin: Parts of the chromosome that remain condensed, genetically inactive, lack genes or genes are repressed, replicates later in the S phase. Eukaryotes have this only. Some chromosomes are all heterochromatic. Mammalian Y chromosome. The X chromosome of mammalian females is condensed into an inert Barr body. The telomere is part of the heterochromatic region, maintains the chromosome’s structural integrity. Centromere: Involved in chromosome movement during cell division.
If you translocate a heterochromatin area elsewhere (even a different chromosome) it can cause new adjacent regions to become inert. This is called the position effect. The position of a gene relative to all other genetic material may affect its expression. Euchromatin: Parts of the chromosome that are uncoiled.
Chromosome Banding Differentiates Regions
Chromosome banding techniques: Made possible differential staining along the longitudinal axis of mitotic chromosomes, staining patterns resemble the bands of polytene chromosomes.
C-banding: Heat denature chromosome and then treat with Giemsa stain, only centromeric regions of mitotic chromosome take up stain, identifies the region of heterochromatin.
G – bands: Digest mitotic chromosomes with proteolytic enzyme trypsin, stain with Giemsa, produces differential staining along the chromosome, reflects heterogeneity and complexity, nomenclature derived from this.
Eukaryotic Genomes and Repetitive DNA
Repetitive DNA has various classes of sequences and organization. Most repetitive sequences don’t encode proteins. Many are transcribed and their RNA remodels chromatin.
Multicopy genes are functional genes present in more than one copy, repetitive.
Three Main Categories of Repetitive Sequences
- Heterochromatin found to be associated with centromeres and making up telomeres.
- Tandem repeats of both short and long DNA sequences.
- Transposable sequences that are interspersed throughout the genome of eukaryotes.
Satellite DNA: Nucleotide composition (GC vs AT) of a species is reflected in DNA’s density, measured by sedimentation equilibrium centrifugation. Makes one single peak meaning a single main band of uniform density but will have other peaks of different density. This is satellite DNA. Makes up a variable portion of DNA depending on the species, not in prokaryotes, highly repetitive DNA (that rapidly reanneals), short sequences repeated many times: sequences found in heterochromatic centromeric regions, different molecular composition.
Centromeric DNA Sequences: Centromeres, primary constrictions along eukaryotic chromosomes, separate homologs during mitosis and meiosis. CEN region: Minimal region of the centromere that supports the function of chromosomal segregation, heterochromatic. Here DNA binds a platform of proteins (including kinetochores). All serve the same function even in other chromosomes. Mutations near the 3’ disrupt centromere function, essential to the binding of the spindle fiber. Alphoid family: Satellite DNA sequences in humans, mainly in centromere regions, 170 bp, in tandem arrays to 1 million bp, this repetitive DNA is transcribed and its RNA is used in kinetochore function.
Telomeric DNA Sequences: Telomere, structure that caps the ends of linear eukaryotic chromosomes, heterochromatic, makes ends inert to other chromosomes and enzymes. Telomeric DNA sequences: Short tandem repeats, stabilize the chromosome. All vertebrates have a 5’-TTAGGGG-3’ repeat. These sequences are transcribed and make the RNA product TERRA: Telomere repeat-containing RNA, critical to heterochromatic nature, facilitates methylation of H3K9 histone. Regulates telomerase (enzyme that regulates telomeres).
Telomerase: RNA component is complementary to TERRA which it uses as a template in making telomeric DNA, uses TERRA as an inhibitor and ligase, active in germ-line cells, inactive in somatic cells. When human cancer cells become malignant they activate telomerase to overcome normal senescence with chromosome shortening.
Middle Repetitive Sequences: VNTRs and STRs: Repetitive DNA, while highly repetitive taking up 5% of the genome, recognized by C(low 0)t analysis, non-coding tandemly repeated sequences or noncoding interspersed sequences, no function.
VNTRs: Variable number tandem repeats, 15-100 bp long, found within or between genes, lengths of repeats 1000-20000 bp, clustered into minisatellites.
STRs: Clustered tandem repeats of di-, tri-, tetra-, and pentanucleotides, repeat unit 2, 3, 4, or 5 bp, STR clusters are called microsatellites, 5-50 repeats per cluster.
Repetitive Transposed Sequences: Interspersed individually throughout the genome, not tandemly repeated, short or long, transposable sequences, mobile and can move to different locations on the genome.
SINEs: Short interspersed nuclear elements, less than 500 bp, present 500,000 times, Alu family, 13% of the genome.
LINEs: Long interspersed nuclear elements, less than 6000 bp, present 850,000 times, L1 family, 21% of the genome.
Middle Repetitive Multicopy Genes: Mid repetitive DNA sometimes has functional genes present in multiple, tandemly repeated copies. Example: ribosomal RNA precursors: 28S, 18S, 5.8S. Many copies of the gene that encodes ribosomal RNA. Example: ribosomal 5S RNA. Multiple copies of the genes encoding 5S rRNA are transcribed separately from multiple clusters, histone-coding genes.