1. CHAPTER 24 Genes and Chromosomes
Key topics:
Organization of information in chromosomes
DNA supercoiling
Structure of the chromosome

2. 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

3. 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”

4. How does genes function? Central Dogma: DNA to RNA to Protein.

5. Genes and Chromosomes What is gene? What is chromosome?
One gene-one enzyme.
One gene-one protein (polypeptide).
Genes are segments of DNA that code for polypeptides and 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 called the chromatin

6. 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

7. The Size and Sequence of DNA Molecules in Bacteria and their viruses
Bacteria(E. coli) 4,639, mm mm

8. T2 phage

9. The sizes of E. coli cell and its DNA

10. DNA from a lysed E. coli cell

11. TABLE 24-2 DNA, Gene, and Chromosome Content in Some Genomes11

12. DNA content and C-value paradox

13. 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

14. Eukaryotic genomes have several sequence components
Nonrepetitive DNA: the complexity of the slow component corresponds with its physical size, i.e., unique sequences.
Moderately repetitive DNA:.component with a Cot1/2 of 10-2 and 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 Usually forms discrete clusters.

15. Types of sequences in the human genome

16. 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

17. Many eukaryotic genes contain intervening sequences (introns)

18. 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

19. Transposons DNA sequence is not completely static
Some sequences, called transposons, can move around within the genome of a single cell
The ends of transposons contain terminal repeats that hybridize with the complementary regions of the target DNA during insertion
To be covered in Ch. 25.

20. Eukaryotic Chromosomes

21. 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)

22. Centromere: Mitotic segregation of chromosomes
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).

23. 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)

24. How is DNA packed in the chromosomes
DNA Supercoiling.
Proteins assisted packaging (nucleosomes)

25. 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 sequence
There are several levels of organization, one of which is the supercoiling of the double-stranded DNA helix

26. Supercoils

27. 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.

28. Negative and positive supercoils .
Topoisomerases catalyze changes in the linking number of DNA.

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

30. The Effects of Replication and Transcription on DNA Supercoiling
FIGURE 24–11 The effects of replication and transcription on DNA supercoiling. Because DNA is a double-helical structure, strand separation leads to added stress and supercoiling if the DNA is constrained (not free to rotate) ahead of the strand separation. (a) The general effect can be illustrated by twisting two strands of a rubber band about each other to form a double helix. If one end is constrained, separating the two strands at the other end will lead to twisting. (b) In a DNA molecule, the progress of a DNA polymerase or RNA polymerase (as shown here) along the DNA involves separation of the strands. As a result, the DNA becomes overwound ahead of the enzyme (upstream) and underwound behind it (downstream). Red arrows indicate the direction of winding. 30

31. Relaxed and supercoiled plasmid DNAs

32. Most cellular DNA is underwound
Normal B-form, relaxed DNA: 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 easier
Linear DNA is underwound with the help of proteins to prevent strands from rotating

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

34. 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.

35. Linking number (Lk) describes supercoiling
In circular DNA, changing the helical turns requires breaking 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.

37. Superhelical Density, 

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

40. Promotion of cruciform structures by DNA underwinding

41. 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

43. Mechanism of Type I topoisomerase action

46. Proposed mechanism of Type II topoisomerase action

47. Topoisomerases are Targets for Antibiotics and Anti-cancer Drugs
Bacterial topoisomerase
inhibitors
Type I topoisomerase
inhibitors 47

48. Human Type II topoisomerase inhibitors48

49. 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.

50. Plectonemic supercoiling

51. DNA Compaction Requires Solenoidal Supercoiling, not plectonemic supercoiling.

52. Changes in Chromosome Structure During the Cell Cycle
FIGURE 24–24 Changes in chromosome structure during the eukaryotic cell cycle. The relative lengths of the phases shown here are for convenience only. The duration of each phase varies with cell type and growth conditions (for single-celled organisms) or metabolic state (for multicellular organisms); mitosis is typically the shortest. Cellular DNA is uncondensed throughout interphase, as shown in the cartoons of the
nucleus in the diagram. The interphase period can be divided (see Fig. 12–44) into the G1 (gap) phase; the S (synthesis) phase, when the DNA is replicated; and the G2 phase, throughout which the replicated chromosomes (chromatids) cohere to each another. Mitosis can be divided into four stages. The DNA undergoes condensation in prophase. During metaphase, the condensed chromosomes line up in pairs along the plane halfway between the spindle poles. The two chromosomes of each pair are linked to different spindle poles via microtubules that extend between the spindle and the centromere. The sister chromatids separate at anaphase, each drawn toward the spindle pole to which it is connected. The process is completed in telophase. After cell division is complete, the chromosomes decondense and the cycle begins anew. 52

53. Changes in chromosome structure during the cell cycle

54. Protein-assisted Packaging of DNA
Nucleosomes are the fundamental organizational units of eukaryotic chromatin

55. 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.

56. DNA wrapped around a nucleosome core

57. DNA Wrapped Around a Histone Core
FIGURE 24–26a DNA wrapped around a histone core. (a) The simplified structure of a nucleosome octamer (left) with DNA wrapped around the histone core (right). 57

58. Front and Side Views of Histone Amino-Terminal Tails
FIGURE 24–26d DNA wrapped around a histone core. (d) Two views of histone amino-terminal tails protruding from between the two DNA duplexes that supercoil around the nucleosome. Some tails pass between the supercoils through holes formed by alignment of the minor grooves of adjacent helices. The H3 and H2B tails emerge between the two coils of DNA wrapped around the histone; the H4 and H2A tails emerge between adjacent histone subunits. 58

59. Histones are small, basic protein
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.

60. 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

61. Effect of DNA Sequence on Nucleosome Binding
FIGURE 24–28 The effect of DNA sequence on nucleosome binding. (PDB ID 1AOI) Runs of two or more A=T base pairs facilitate the bending of DNA, while runs of two or more G≡C base pairs have the opposite effect. When spaced at about 10 bp intervals, consecutive A=T base pairs help bend DNA into a circle. When consecutive G≡C base pairs are spaced 10 bp apart, and offset by 5 bp from runs of A=T base pairs, DNA binding to the chromosome is facilitated. 61

62. Chromatin assembly

63. Nucleosomes are packed into successively higher-order structures
The 30 nm fiber, a higher-order organization of nucleosomes.

64. The 30 nm Fiber
FIGURE 24–29 The 30 nm fiber, a higher-order organization of nucleosomes. The compact fiber is formed by the tight packing of nucleosomes. (a) The 30 nm fiber, as seen by electron microscopy. There are two proposed models for the structure that are consistent with available data: (b) the solenoid model featuring one helical array of nucleosomes and (c) the zigzag model featuring two helical arrays of nucleosomes wrapped about each other. The black line is intended only to trace the proposed general path of the organized structure. 64

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

66. Loops of DNA Attached to a Chromosomal Scaffold
FIGURE 24–30 Loops of DNA attached to a chromosomal scaffold. (a) A swollen chromosome, produced in a buffer of low ionic strength, as seen in the electron microscope. Notice the appearance of 30 nm fibers (chromatin loops) at the margins. (b) Extraction of the histones leaves a proteinaceous chromosomal scaffold surrounded by naked DNA. (c) The DNA appears to be organized in loops attached at their base to the scaffold in the upper left corner. Scale bar = 1 µm. The three images are at different magnifications. 66

67. Higher order of folding is not yet understood
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.

68. Compaction of DNA in a Eukaryotic Chromosome
FIGURE 24–31 Compaction of DNA in a eukaryotic chromosome. This model shows the levels of organization that could provide the observed degree of DNA compaction in the chromosomes of eukaryotes. First the DNA is wrapped around histone octamers, then H1 stimulates formation of the 30 nm fiber. Further levels of organization are not well understood but seem to involve further coiling and loops in the form of rosettes, which also coil into thicker structures. Overall, progressive levels of organization take the form of coils upon coils upon coils. It should be noted that in cells, the higher-order structures (above the 30 nm fiber) are unlikely to be as uniform as depicted here. 68

69. Model of DNA compaction in eukaryotic chromosomes

70. Condensed chromosome are maintained by SMC proteins

71. Structure of SMC Proteins
FIGURE 24–32 Structure of SMC proteins. (a) SMC proteins have five domains. (b) Each SMC polypeptide is folded so that the two coiled-coil domains wrap around each other and the N and C domains come together to form a complete ATP-binding site. Two polypeptides are linked at the hinge region to form the dimeric V-shaped SMC molecule. (c) Bacterial SMC proteins form a homodimer. The six different eukaryotic SMC proteins form heterodimers. Cohesins are made up of SMC1-SMC3 pairs, and condensins consist of SMC2-SMC4 pairs. The SMC5-SMC6 pair is involved in DNA repair. (d) Electron micrographs of SMC dimers from the bacterium Bacillus subtilis. 71

73. Model for the effect of condensins on DNA supercoiling

74. Possible Role of Condensins
FIGURE 24–33 The possible role of condensins in chromatin condensation. Initially, the DNA is bound at the hinge region of the SMC protein, in the interior of what can become an intramolecular SMC ring. ATP binding leads to head-to-head association, forming supercoiled loops in the bound DNA. Subsequent rearrangement of the head-to-head interactions to form rosettes condenses the DNA. Condensins may organize the looping of the chromosome segments in a number of ways. Two current models are shown. 74

75. Bacterial DNA is organized into nucleoids
Can occupy much of cell volume
DNA attaches to plasma membrane
Scaffold-like structure organizes the circular DNA into ~500 looped domains
DNA binds to proteins transiently
Example: Protein HU

76. Looped Domains of the E. coli Chromosome
FIGURE 24–36 Looped domains of the E. coli chromosome. Each domain is about 10,000 bp in length. The domains are not static, but move along the DNA as replication proceeds. Barriers at the boundaries of the domains, of unknown composition, prevent the relaxation of DNA beyond the boundaries of the domain where a strand break occurs. The putative boundary complexes are shown as gray-shaded ovoids. The arrows denote movement of DNA through the boundary complexes. 76