Lecture 3: Bacteria and Eukaryotes Chromosomes PDF

Lecture#3 Bacteria and eukaryotes chromasomes - Introduction Chromosomes are responsible for physically carrying the DNA and all associated proteins. Even the tiniest of beings, bacteria, can hold anywhere between 130 kbp and 14 Mbp of DNA, while the human genome holds over 3 billion bps w...

Lecture#3 Bacteria and eukaryotes chromasomes - Introduction Chromosomes are responsible for physically carrying the DNA and all associated proteins. Even the tiniest of beings, bacteria, can hold anywhere between 130 kbp and 14 Mbp of DNA, while the human genome holds over 3 billion bps which would measure ~2 m if spread. There are many variations in the form of DNA packaging observed in different life forms. E. coli genome comprises of a single DNA molecule which is present in the form of a single circular covalently closed chromosome and compacted with the help of packaging proteins. On the other hand, the haploid human genome has its DNA segregated into 23 units that form morphologically distinct linear chromosomes on compaction (Fig. 8.2). Fig. 8.2 From: Chromosomal Organization of DNA Varying forms of chromosomes. (a) The diagram is a schematic representation of a bacterial nucleoid. It depicts the bottle brush model of the nucleoid with supercoiled loops that are interwound and radiate out of a dense core. (b) A schematic SKY image of a normal female karyotype 11  - Function structure relations of chromasomes However, chromosomes universally perform two fundamental functions: the precise transmission of genetic information and accurate control of gene expression. Specialized regions containing repetitive sequences help form structures such as telomeres, replication origin, etc. which help the chromosomes in the execution of its functions. The level of chromosome compaction also varies depending on which areas are needed for active transcription and which areas will remain inactive. Our current ideas of chromosomal structures revolve around understanding how chromatin is arranged in structural domain in the three-dimensional nuclear spaces and how this structural landscaping controls different facets of gene expression. - DNA Supercoiling The genetic material that must be packaged into a comparatively miniscule cellular space, it stands to reason that the DNA strings must be highly coiled and condensed. Yet, the coiling should still leave functionally important domains of the DNA accessible to proteins. This feat is achieved through the process of supercoiling which literally implies the coiling of a coil. 21 To understand the process, let us visualize the double helical DNA. An axis passes through the middle of the helix. When this axis is folded upon itself, it results in a supercoiled DNA (Fig. 8.3). This can be explained by using a small linear double helix with two to three turns. If both ends of the helix are twisted in direction of the helix winding, the number of turns in the helix will seem to increase. This supercoiled helix is over-twisted and under strain. If, however, the helix is twisted in the direction opposite to its coiling, it will appear to unwind. This state of the coil with lesser number of turns is a relaxed state. If the molecule is consistently over-twisted, it will relieve the molecular strain by twisting upon its own helical axis, thus creating a positive supercoil. Likewise an under-twisted moiety will result in a negative supercoil. Most basic processes like replication and transcription require the unwinding of the double helix. This is turn results in over-twisting of the domains lying ahead. Thus, supercoiling is an integral aspect of the tertiary structure of the DNA that is omnipotent in cellular DNA and tightly regulated by the cell. Fig. 8.3 From: Chromosomal Organization of DNA 31 The supercoiling of DNA. A linear double helix is shown on the left-hand side with an imaginary axis passing through it. The right-hand figure depicts a supercoiled DNA where the axis has folded on itself To understand the physiological relevance of supercoiling, let us focus on closed circular B-form of DNA which has 10.5 bps per turn (Fig. 8.4). The underwinding of this DNA at any point will result in a strain. For example, if the DNA has 84 bps, in a relaxed state, it will consist of eight helical turns of 10.5 bps each. The DNA is now underwound, and one of these eight turns is removed, and then the 84 bps will be divided in seven helical turns containing 12 bps per turn instead of the 10.5 bps. This alteration will result in a thermodynamically strained structure. This strain can be compensated in two ways. Either the strands can simply separate over a stretch to resort to 10.5 bps per turn or the axis of the double helix can coil on itself to realign the base stacking to approximate of 10.5 bps per turn pattern. Fig. 8.4 From: Chromosomal Organization of DNA 41 DNA underwinding. (a) A relaxed 84 bps DNA segment with eight helical turns. (b) The underwinding of the DNA by removing one turn results in a strained DNA which is compensated by (c) separation of the strand over 10.5 bps or (d) by forming a supercoil. - Organization of the Prokaryotic Chromosome Having understood the basic dynamics of DNA supercoiling, we move onto the question of how the DNA is packaged in prokaryotes lacking a defined nucleus and how the stability of the coiled structures are maintained. Most of our knowledge of the organization of prokaryotic chromosomes comes from the studies conducted on E. coli. The single circular chromosome is packaged in the form of a nucleoid which is defined as the area of the cell where the chromosome DNA is located (Fig. 8.7). Fig. 8.7 51 Packaging of the bacterial chromosome. (a) A circular chromosome without any compaction. (b) The DNA-associated proteins form a central scaffold, and the bacterial DNA loops around them. (c) The loops are further supercoiled to form a condensed structure Full size image The packaging of the bacterial genome into the small nucleoid space occurs via two processes; the first is the process of supercoiling discussed in Sect. 8.2, and the other is the interaction of the DNA with packaging proteins. The main histone-like packaging proteins present in the E. coli are HU, IHF (integration host factor), Fis (factor for inversion stimulation) and H-NS. These positively charged proteins interact with the negatively charged DNA and, together with the topoisomerase and gyrase enzymes, maintain the supercoiling homeostasis of the bacterial genome. During normal growth most of the bacterial genome is negatively supercoiled. 61 Proteins associated with bacterial chromosomes. Several proteins like HU and H-NS are associated with the bacterial DNA to maintain its topological structure - Hierarchical Packaging of the Eukaryotic Chromosome When DNA from eukaryotic cells is isolated, it is found to be associated with nearly equivalent proportions of protein in an extremely compacted complex called chromatin. Even so, the chromosome has specialized structures, and its dynamic topology allows access to proteins required for life functions. This is achieved by a hierarchical and orchestrated process of packaging with the help of different types of proteins (Fig. 8.9). Fig. 8.9 71 The hierarchical packaging of eukaryotic chromosome. A simple dsDNA wounds around a nucleosome complex giving rise to a beads on string structure. This nucleosome forms a solenoid complex of 30 nm diameter. The 30 nm fibre further forms 300 nm long loops around the non-histone proteins undergoing several levels of compaction to form the chromosome. 4.1 The Nucleosome Assembly The most predominant proteins found attached to the eukaryotic DNA are the histone proteins. They represent a family of basic proteins, rich in positively charged amino acids such as lysine and arginine. This positive charge helps the protein interact with the negatively charged backbone of DNA. There are five principal types of histones: H1, H2A, H2B, H3 and H4. The histones form a highly structured assembly, and the DNA loops around 81 it to give rise to the beads on strings moieties known as the nucleosome assembly. The nucleosome consists of a string of DNA wrapped around a protein core like a thread on a spool. The disc-shaped core is an octamer comprising of two copies of H2A, H2B, H3 and H4 each. Histone protein sub-assemblies come together to form the histone core with the H3 and H4 forming a tetrameric sub-assembly and the H2A-H2B dimeric sub-assembly joining it to complete the histone core. The DNA wraps around this core ~1.65 times using around 146 bps length. The H3 and H4 tetramers interact in the middle and the rear ends, while the rest of the DNA is bound to the H2A–H2B dimer via hydrogen bonds. Consecutive nucleosome cores are connected by short segments of linker DNA which harbours the linker histone, H1 (Figs. 8.10and 8.11). Fig. 8.10 An electron micrograph of nucleosome “beads-on-string” structure. The black brackets indicate nucleosome assembly, black arrowheads indicate the nucleosomal core, and white arrowheads indicate linker DNA. The scale bar indicates 50 nm. Image credit: Chris Woodcock Fig. 8.11 91 The nucleosome assembly. The nucleosome consists of an octameric core comprised of the H2A, H2B, H3 and H4 units with about 146 bps of DNA wound around it - The Heterochromatin and Euchromatin As discussed earlier, one of the most important tasks of the chromatin and chromosomal structures is to allow the accurate control of gene expression. Therefore, the chromatin fibre must make allowances for the proteins to reach and transcribe a segment of DNA required to be active while leaving other non-relevant segments inactive. In most regions the chromatin appears to be less compactly packaged and relatively more dispersed in the nucleus. These regions are transcriptionally more active and are known as the euchromatin. On the other hand, the more densely packaged regions of chromatin resembling the level of condensation seen in the chromosomes are transcriptionally inactive and known as the heterochromatin. 101 7.3 Satellite DNA The satellite DNA consists of simple or moderately complex DNA sequence repeated multiple times over a long stretch of the DNA in tandem (end to end). Satellite DNA repeats do not code for proteins. However, biologists hypothesized they might have various biological roles, such as in regulating recombination, chromosome segregation, gene expression, structural roles, or roles in embryonic development 7.4 The Telomere A distinctive feature of the eukaryotic chromosome is the telomeric region present at the end of each chromatid arm. They form sticky ends when the chromosomes are broken and differ markedly in structure and function from the remainder of the chromosome. A span of 10–15 kbps region of highly conserved hexameric repeat (TTAGG)n makes up the telomere (Fig. 8.19). The main functions of a telomere are to maintain chromosomal stability and prevent chromosomal degradation. Fig. 8.19 The telomeric sequence of various organisms. The sequence of telomerase RNA and the telomere repeat for different organisms have been depicted. 111 QUESTIONS - CHOOSE THE CORRECT ANSWERA 1. What are the two fundamental functions universally performed by chromosomes? A) Energy production and cell division B) Genetic transmission and gene expression control C) Protein synthesis and cell differentiation D) Cell signaling and waste elimination 2. Which of the following specialized regions help chromosomes in executing their functions? A) Centrioles and mitochondria B) Telomeres and replication origin C) Golgi apparatus and endoplasmic reticulum D) Ribosomes and lysosomes 3. What is the physiological relevance of DNA supercoiling? A) It helps in energy production B) It is essential for cell division C) It maintains the tertiary structure of DNA D) It regulates protein synthesis 4. How is the DNA packaged in prokaryotes like E. coli? A) In the form of linear chromosomes B) Via histone proteins C) Into a nucleoid structure D) With the help of nucleosomes 5. What is the main function of histone proteins in eukaryotic cells? A) Energy storage 121  B) DNA replication C) Structural organization of chromosomes D) Protein synthesis 6. Which type of proteins interact with the negatively charged DNA in prokaryotes to maintain supercoiling homeostasis? A) Histones B) Topoisomerases C) Gyrase enzymes D) HU, IHF, Fis, and H-NS proteins 7. What is the basic structural unit of chromatin in eukaryotic cells? A) Nucleosome B) Ribosome C) Mitochondrion D) Golgi apparatus 8. What is the composition of a nucleosome core? A) Octamer of histone proteins B) Linear DNA strand C) Lipid bilayer D) RNA polymerase 131