Prokaryotic cells do not contain nuclei or other membrane-bound organelles. The nucleoid is the area of a prokaryotic cell in which the chromosomal DNA is located. Chromosome is several orders of magnitude larger than the cell itself. So, if bacterial chromosomes are so huge, how can they fit comfortably inside a cell—much less in one small corner of the cell? Most prokaryotes do not have histones (except some species of Archaea). Thus, one way prokaryotes compress their DNA into smaller spaces is through supercoiling. Most bacterial genomes are negatively supercoiled during normal growth. Multiple proteins act together to fold and condense prokaryotic DNA. One most abundant protein HU, found in the nucleoid, works with topoisomerase I to bind DNA and introduce sharp bends in the chromosome, Generating the tension necessary for negative supercoiling. Recent studies… other proteins like integration host factor (IHF), can bind to specific sequences within the genome and introduce additional bends. The folded DNA is then organized into a variety of conformations that are supercoiled and wound around tetramers of the HU protein, much like eukaryotic chromosomes are wrapped around histones.

1. Genome Organization:
Prokaryotic genome organization

2. • Prokaryotic cells do not contain nuclei or other
membrane-bound organelles.
• The nucleoid is the area of a prokaryotic cell in
which the chromosomal DNA is located.
• Chromosome is several orders of magnitude
larger than the cell itself.
• So, if bacterial chromosomes are so huge, how
can they fit comfortably inside a cell—much
less in one small corner of the cell?

3. • Most prokaryotes do not have histones
(except some species of Archaea).
• Thus, one way prokaryotes compress their
DNA into smaller spaces is through
supercoiling.

4. Figure 8.1 Genomes 3 (© Garland Science 2007)
No nuclear envelope. Instead, a nucleoid

6. Figure 8.2 Genomes 3 (© Garland Science 2007)
Genome can be naturally
compacted by supercoiling it

7. Figure 8.3 Genomes 3 (© Garland Science 2007)
Model for genome organization

9. Most bacterial genomes are negatively
supercoiled during normal growth.
• Multiple proteins act together to fold and
condense prokaryotic DNA.
• One most abundant protein HU, found in the
nucleoid, works with topoisomerase I to bind
DNA and introduce sharp bends in the
chromosome, Generating the tension
necessary for negative supercoiling.

10. • Recent studies… other proteins like integration
host factor (IHF), can bind to specific
sequences within the genome and introduce
additional bends.
• The folded DNA is then organized into a variety
of conformations that are supercoiled and
wound around tetramers of the HU protein,
much like eukaryotic chromosomes are
wrapped around histones.

11. • Bacterial DNA binding Protein

12. Once the prokaryotic genome has been
condensed, DNA topoisomerase I, DNA
gyrase, and other proteins help maintain the
supercoils.
One of these maintenance proteins,
histone-like nucleoid-structuring (H-NS),
plays an active role in transcription by
modulating the expression of the genes
involved in the response to environmental
stimuli.

14. Figure 2: A conserved geometry for transcription initiation in eukaryotes and
bacteria. The illustration compares the binding of RNA polymerase to an apical
loop in a bacterial plectoneme with a similar binding of a polymerase-initiation
complex to a short plectoneme generated by the removal of two nucleosomes.
These plectonemic structures would be stabilized by HU in bacteria and high-
mobility group box (HMGB) proteins in eukaryotes. The proteins are shown
binding to crossovers but would also probably bend the interwindings. TFIID,
basal transcription factor.

15. Eukaryotic genome organization

17. • The genomic DNA of eukaryotes is very long
(about 2 m in humans).
• Packaging of the genome involves coiling of
the DNA in a left-handed spiral around
molecular spools, made of histone octamers,
to form nucleosomes.
• About 80% of the genomic DNA is organized
as nucleosomes.
• The histone octamer reveals a tripartite
structure, organized into the central (H3/H4)2
tetramer and two peripheral H2A/H2B dimers.

18. • Nucleosome assembly is initiated by
wrapping a 121 bp DNA segment around a
tetramer of histones (H3/H4)2.
• Association of H2A/H2B dimers at either side
of the tetramer organizes 147 bp of DNA.
DNA is a moderately flexible polymer with a
persistence length of about 150 bp

19. Nucleosome structure
Nucleosome core particle:
octamer of histones plus
~146 bp DNA
Octamer of histones plus
~146 bp DNA AND linker
histone H1

20. • In the absence of exogenous forces,
150 bp of DNA essentially follow a
straight path,
but in a nucleosome, it coils in 1.65
toroidal superhelical turns around the
octamer and thus is severely
distorted…..????

21. • DNA bending around the
nucleosome is expected to
happen at high energy
costs!!!!!!!!!!!!!!!!!!!!!!!!!!!!

22. • Energy cost is compensated by
• DNA histone interactions occurring
approximately every 10 bp on each
DNA strand, generating 7 histone–DNA
interaction clusters per DNA coil
(superhelical locations (SHL) .

23. • The DNA–Histone interactions are
stabilized by more than 116 direct and
358 water-bridged interactions,
rendering the Nucleosome a stable
particle in the absence of additional
factors.
• https://www.youtube.com/watch?v=gbS
IBhFwQ4s
https://www.youtube.com/watch?v=DcDh
L95PaRU
https://www.youtube.com/watch?v=X_tYr
nv_o6A

27. Chromatin packaging hierarchy
Level 1:
nucleosome formation
Level 2:
30 nm fiber
Level 3:
Nuclear scaffolding
Level 4:
Mitotic (metaphase)
chromosome

28. Level Two: the 30nm fiber
• Requires Histone H1
• Compaction ratio approx 100 fold
Lehninger

29. Level three: nuclear
scaffolding
• Not well understood
• Organization is not random; involved sequence
elements (red dots), more non-histone chromatin
proteins and tethering to the nuclear envelope and
matrix

30. Genome contortions during
the cell cycle
Time for replication,
transcription
Time for cell division:
no gene expression

31. Metaphase chromatin:
level 4 packaging: fully condensed

32. Interphase chromatin: levels 1-3
relatively decondensed chromosomes
• Heterochromatin:
dark-staining,
condensed (mostly
simple-sequence
DNA)
• Euchromatin: light-
staining, less
condensed
(complex sequence
DNA: e.g. genes)

33. Heterochromatin vs Euchromatin
• Stains darkly (highly
condensed)
• Repetitive sequences
• Replicates later in the
cell cycle
• Little or no
recombination
• Transcriptionally
repressive: silences
gene expression
• Stains lightly
(decondensed)
• Single copy
sequences (genes)
• Replicates early in the
cell cycle
• Recombines
• Transcriptionally
active: permissive
for gene expression

34. Chromosome Structure Regulation
• Two types of mechanisms to regulate
chromatin structure;
1.Histone modifiers (enzymes)
• Modify histone tail residues, help in
recruitment of other factors, do not
change position of the nucleosome
2. Chromatin remodelers (complexes)
–Change the position of nucleosomes (ATP
dependent)
–Why modification and remodeling? 34

35. • Histones are subjected to a variety of post
translational modifications (most often on
the N-terminal tails) may be covalent
modifications
• These modifications are generated by
specific enzymes
• These modifications are recognized by
proteins that can influence gene expression
and other chromatin functions

36. • Acetylated N-terminal histone tails bind
DNA with reduced affinity and are more
mobile with respect to the DNA surface
than unmodified tails.
• Acetylation disrupts the secondary
structures that are known to exist within
the H3 and H4 N-termini when they are
bound to nucleosomal DNA. This might
further destabilize interactions with DNA
and the nucleosome itself.

37. • Beyond effects on individual
nucleosomes, acetylation facilitates
factor access and transcription from
nucleosomal arrays by decreasing the
stability of the completely compacted
30-nm fiber

38. • Chromatin modifications:
• Histone code??
• A hypothesis that the transcription of genetic
information encoded in DNA is in part
regulated by chemical modifications to histone
proteins
38
Modification Proposed function
Acetylation Transcription activation, Histone deposition, DNA repair,
chromosome assembly
Methylation Transcription activation/ silencing, checkpoint response
Phosphorylation DNA repair, Apoptosis, Mitosis, Transcriptional activation
Ubiquitylation Spermatogenesis, Meiosis, Transcriptional activation
Sumoylation Transcriptional repression
Biotinylation Gene expression

39. Chromatin Modifications
39
Acetyle Phosphoryle
Methyl
Ubiquitin and
SUMOyl

40. Histone tails play important role in
chromatin modification and remodeling
40

43. • Enzymes that establish a mark on either DNA or the histone tail
are termed 'writers'. These modifications can be removed or
modified by 'editing' enzymes. The third class of enzymes
includes the 'readers' of an epigenetic mark, which mediate the
interaction of the mark with a protein complex to exert effects on
transcription.
• The top panel depicts DNA modifications, such as DNA
methylation and demethylation, and the enzymes involved; the
bottom panel shows histone modifications and the enzymes
involved. Examples for each class of enzyme are given. 5hmC,
5-hydroxymethylcytosine; 5mC, 5-methylcytosine; BAZ1B,
tyrosine protein kinase BAZ1B; BRCT, BRCT domain-
containing protein; CHD, chromodomain helicase DNA-binding
protein; DIDO1, death-inducer obliterator 1; DNMT, DNA
methyltransferase; HAT, histone acetyltransferase; HDAC,
histone deacetylase; HMT, histone methyltransferase; KDM,
lysine-specific histone demethylase; MECP2, methyl-CpG-
binding protein 2; PPP, serine/threonine protein phosphatase;

44. Chromatin Remodeling
• Chromatin remodeling complexes use the energy
of ATP hydrolysis to displace and reposition nucleosomes,
thereby altering chromatin accessibility.
44

45. 45

46. Classifying chromatin
remodelers
• Chromatin remodeling complexes are
classified based on
• protein motifs found in addition to the
ATPase domain,
• or on how the ATPase domain itself is
structured
46

47. SWI2/SNF2
ATPase
SUPERFAMILY
SWI2/SNF2
subfamily
ISWI
subfamily
CHD/Mi2
subfamily
Ino80
subfamily
ATPase BROMO
ATPase SANT
ATPase
DNA binding
CHROMO
ATPase ATPase
SANT
SWI3 ADA2 N-CoR TFIIIB
Originally isolated
genetically in
budding yeast as
mutants in mating
type switching and
sucrose non-
fermenting
functions
BROMO
from Drosophila: Brahma

48. Chromatin remodeling complexes
hBrm
SWI2/SNF2 subfamily ISWI subfamily

49. From Clapier and Cairns, Annu. Rev. Biochem. 2009
Shared characteristics of chromatin
remodeling complexes
• bind nucleosomes
• are DNA-dependent ATPases
• recognize histone modifications
• ATPase activity can be regulated
• interact with other proteins

50. Chromatin Remodeling. Eukaryotic gene regulation begins with an
activated transcription factor bound to a specific site on DNA. One
scheme for the initiation of transcription by RNA polymerase II requires
five steps: (1) recruitment of a coactivator, (2) acetylation of lysine
residues in the histone tails, (3) binding of a remodeling engine complex
to the acetylated lysine residues, (4) ATP-dependent remodeling of the
chromatin structure to expose a binding site for RNA polymerase or for
other factors, and (5) recruitment of RNA polymerase. Only two subunits
are shown for each complex, although the actual complexes are much
larger. Other schemes are also possible.

51. Mechanism of Remodeling
51

52. 52
Histone Acetyle
transferase

53. 53

54. 54

55. 55
Transcription complex

56. 56

57. Some Techniques Used
• Mutational Analysis (S. cerivisae)
SWI/SNF complex
• Micrococcal Nuclease Digestion
• X-Ray studies (NCPs)
• Electron Microscopy
• Bioinformatics tools
57

58. Summary
• Structural organization of chromosomes varies in
prokaryotes and eukaryotes with much more
complexity in eukaryotes
• DNA is highly packaged inside cell in order to fit
into small volume
• Nucleosome is the defined structural unit of
eukaryotic chromosome
• Chromatin modification and Remodeling play
important roles in regulation of expression of
genes not easily accessible by cellular machinery
• Mutations in chromatin regulatory elements can
have severe effects
• SWI/SNF is the most studied remodeling complex
58

59. Assisted readings
• Sebastian Glatt*, Claudio Alfieri* and
Christoph W Mu¨ ller. Recognizing and
remodeling the nucleosome. Current
Opinion in Structural Biology 2011,
21:335–341
• Genes X, Lewin, (Chapter 10)
• Biology, 7th edition , Raven
• Genes VIII, Lewin
59