Post on 02-Jun-2018
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STRUKTUR DAN FUNGSI BIOMOLEKUL
KOMPLEKS / SUPRAMOLEKUL:
MEMBRAN, KROMOSOM, RIBOSOM
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Supramolecular chemistry
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Supramolecular chemistry
"Supramolecular chemistry is the chemistry of the intermolecular
bond, covering the structures and functions of the entities formedby the association of two or more chemical species"
J.-M- Lehn
"Supramolecular chemistry is defined as chemistry "beyond themolecule", as chemistry of tailor-shaped inter-molecular
interaction. In 'supramolecules' information is stored in the form of
structural peculiarities. Moreover, not only the combined action of
molecules is called supramolecular , but also the combined action
of characteristic parts of one and the same molecule.
F. Vögtle
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Supramolecular chemistry – in nature
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Supramolecular chemistry – in nature
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Supramolecular chemistry – in nature
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Supramolecular chemistry – in nature
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The organic compounds from which most
cellular materials are constructed
• Amino Acids
• Nucleotides
• Carbohydrates
• Lipids
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Structural hierarchy in the molecular
organization of cells
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MEMBRANES
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Membrane Structure
The fluid mosaic model of membrane structure
contends that membranes consist of:
-phospholipids arranged in a bilayer
-globular proteins inserted in the lipid bilayer
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Membrane Structure
Cellular membranes have 4 components:
1. phospholipid bilayer
2. transmembrane proteins3. interior protein network
4. cell surface markers
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Membrane Structure
Membrane structure is visible using an electron
microscope.
Transmission electron microscopes (TEM) can
show the 2 layers of a membrane.
Freeze-fracturing techniques separate the layers
and reveal membrane proteins.
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Phospholipids
Phospholipid structure consists of
-glycerol – a 3-carbon polyalcohol acting as abackbone for the phospholipid
-2 fatty acids attached to the glycerol
-phosphate group attached to the glycerol
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Phospholipids
The partially hydrophilic, partially
hydrophobic phospholipid spontaneously
forms a bilayer:-fatty acids are on the inside
-phosphate groups are on both surfaces
of the bilayer
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Phospholipids
Phospholipid bilayers are fluid.
-hydrogen bonding of water holds the 2 layerstogether
-individual phospholipids and unanchoredproteins can move through the membrane
-saturated fatty acids make the membrane
less fluid than unsaturated fatty acids-warm temperatures make the membranemore fluid than cold temperatures
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Phospholipids
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Membrane Proteins
Membrane proteins have various functions:
1. transporters
2. enzymes3. cell surface receptors
4. cell surface identity markers
5. cell-to-cell adhesion proteins6. attachments to the cytoskeleton
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Membrane Proteins
Peripheral membrane proteins
-anchored to a phospholipid in one layer of
the membrane
-possess nonpolar regions that are inserted in
the lipid bilayer
-are free to move throughout one layer of the
bilayer
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Membrane Proteins
Integral membrane proteins
-span the lipid bilayer (transmembrane
proteins)
-nonpolar regions of the protein are
embedded in the interior of the bilayer
-polar regions of the protein protrude from
both sides of the bilayer
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Membrane Proteins
Integral proteins possess at least one
transmembrane domain
-region of the protein containing hydrophobic
amino acids
-spans the lipid bilayer
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Membrane Proteins
Extensive nonpolar regions within a
transmembrane protein can create a pore
through the membrane.
-b sheets in the protein secondary structure
form a cylinder called a -barrel
-b-barrel interior is polar and allows water and
small polar molecules to pass through the
membrane
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Passive Transport
Passive transport is movement of molecules
through the membrane in which
-no energy is required
-molecules move in response to a
concentration gradient
Diffusion is movement of molecules from high
concentration to low concentration
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Passive Transport
Channel proteins include:
-ion channels allow the passage of ions
(charged atoms or molecules) which are
associated with water
-gated channels are opened or closed in
response to a stimulus
-the stimulus may be chemical or electrical
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Passive Transport
Carrier proteins bind to the molecule that theytransport across the membrane.
Facilitated diffusion is movement of a moleculefrom high to low concentration with the helpof a carrier protein.
-is specific
-is passive
-saturates when all carriers are occupied
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Passive Transport
When 2 solutions have different osmotic
concentrations
-the hypertonic solution has a higher solute
concentration
-the hypotonic solution has a lower solute
concentration
Osmosis moves water through aquaporins
toward the hypertonic solution.
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Passive Transport
Organisms can maintain osmotic balance indifferent ways.
1. Some cells use extrusion in which water is
ejected through contractile vacuoles.2. Isosmotic regulation involves keeping cellsisotonic with their environment.
3. Plant cells use turgor pressure to push thecell membrane against the cell wall and keepthe cell rigid.
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Active Transport
Active transport
-requires energy – ATP is used directly orindirectly to fuel active transport
-moves substances from low to high
concentration
-requires the use of carrier proteins
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Active Transport
Carrier proteins used in active transport include:
-uniporters – move one molecule at a time
-symporters – move two molecules in thesame direction
-antiporters – move two molecules inopposite directions
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Active Transport
Sodium-potassium (Na+-K+) pump
-an active transport mechanism
-uses an antiporter to move 3 Na+ out of the
cell and 2 K+ into the cell-ATP energy is used to change theconformation of the carrier protein
-the affinity of the carrier protein for eitherNa+ or K+ changes so the ions can be carriedacross the membrane
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Active Transport
Coupled transport
-uses the energy released when a molecule
moves by diffusion to supply energy to active
transport of a different molecule
-a symporter is used
-glucose-Na+ symporter captures the energy
from Na+ diffusion to move glucose against a
concentration gradient
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Bulk Transport
Endocytosis occurs when the plasma membraneenvelops food particles and liquids.
1. phagocytosis – the cell takes in particulate
matter2. pinocytosis – the cell takes in only fluid
3. receptor-mediated endocytosis – specific
molecules are taken in after they bind to areceptor
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Modification of Cell Surfaces
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Cell Surfaces in Animals
•Junctions Between Cells are points of contact between cells that allow
them to behave in a coordinated manner.•Anchoring junctions mechanically attach adjacent cells.•In adhesion junctions, internal cytoplasmic plaques, firmly attachedto cytoskeleton within each cell are joined by intercellular filaments;
they hold cells together where tissues stretch (e.g., in heart, stomach,
bladder).
•In desmosomes, a single point of attachment between adjacent cellsconnects the cytoskeletons of adjacent cells.
•In tight junctions, plasma membrane proteins attach in zipper-likefastenings; they hold cells together so tightly that the tissues are
barriers (e.g., epithelial lining of stomach, kidney tubules, blood-brain
barrier).
•A gap junction allows cells to communicate; formed when two
identical plasma membrane channels join.•They provide strength to the cells involved and allow themovement of small molecules and ions from the cytoplasm of
one cell to the cytoplasm of the other cell.
•Gap junctions permit flow of ions for heart muscle and smoothmuscle cells to contract.
Modification of Cell Surfaces
Cell-Surface Modifications:
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Junctions
E t ll l M t i
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•The extracellular matrix is a meshwork of polysaccharides and proteins produced byanimal cells.
•Collagen gives the matrix strength and elastin gives it resilience.•Fibronectins and laminins bind to membrane receptors and permitcommunication between matrix and cytoplasm; these proteins also form
“highways” that direct the migration of cells during development. •Proteoglycans are glycoproteins that provide a packing gel that joins thevarious proteins in matrix and most likely regulate signaling proteins that bind to
receptors in the plasma protein.
Extracellular Matrix
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CHROMOSOMES
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Chromosomes:
•Chromosome structure
•Chromatin structure
•Chromosome variations
•“The new cytogenetics”
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Prokaryotic chromosomes
•Circular double helix
•Complexed with protein in astructure termed the nucleoid
•Attached to plasmamembrane
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Eukaryotic Chromosomes
• Located in the nucleus
• Each chromosome consists of a single molecule ofDNA and its associated proteins
The DNA and protein complex found in eukaryoticchromosomes is called chromatin
1/3 DNA and 2/3 protein
•Complex interactions between proteins and nucleicacids in the chromosomes regulate gene and
chromosomal function
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d h h
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• Pulsed-field gel electrophoresis - separation of
chromosomes
• Analysis of the complete nucleotide sequence
of many genomes now
• In situ hybridization (below)
Some evidence that chromosomes contain a
single DNA molecule
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Ideogram
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From Miller & Therman (2001) Human
Chromosomes, Springer
Ideogram
•Diagramatic representation
of a karyotype
•Individual chromsomes arerecognized by
-arm lengths
p, short
q, long
-centromere position
metacentric
sub-metacentricacrocentric
telocentric
-staining (banding) patterns
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Ch b di
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• Q (quinicrine) & G (Giemsa) banding
preferentially stain AT rich regions
• R (reverse banding) preferentially stains GC-rich
regions
•C-banding (denaturation & staining)preferentially stains constitutive
heterochromatin, found in the centromere
regions and distal Yq
Chromsome banding
C-banded karyotype of XY cell
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C banded karyotype of XY cell
From Miller & Therman (2001) Human
Chromosomes, Springer
C tit ti h t h ti
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• Tandem, highly repeated short sequences of
DNA
– Non-coding and non-expressing
– Buoyant density discrete from the bulk of the
genome (satellite DNA )
• C-banding
• Late replicating
• Maintains a highly compacted organization
• Never transcribed
Constitutive heterochromatin
Facultative heterochromatin
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Facultative heterochromatin
• All types of sequences
• C-banding negative
• Late replicating
• Condensed conformation
• Not transcribed
• Includes genes silenced in specific cell typesand/or at specific times in development
• e.g. inactivated X chromosomes
Euchromatin
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• Actively expressed sequences
• More open conformation
Euchromatin
Fluorescence in situ a | The basic elements of fluorescence in situhybridization are a DNA probe and a targetb | B f h b idi ti th DNA b
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hybridization (FISH) sequence. b | Before hybridization, the DNA probeis labelled by various means such as NICKTRANSLATION, RANDOM-PRIMED LABELLING
and PCR. Two labelling strategies are commonly
used — indirect labelling (left panel) and direct
labelling (right panel). For indirect labelling,
probes are labelled with modified nucleotides that
contain a HAPTEN, whereas direct labelling uses
the incorporation of nucleotides that have been
directly modified to contain a fluorophore. c | The
labelled probe and the target DNA are denatured
to yield ssDNA. d | They are then combined,which allows the annealing of complementary
DNA sequences. e | If the probe has been labelled
indirectly, an extra step is required for
visualization of the non-fluorescent hapten that
uses an enzymatic or immunological detection
system. Whereas FISH is faster with directly
labelled probes, indirect labelling offers the
advantage of signal amplification by using several
layers of antibodies, and might therefore produce
a signal that is brighter compared with
background levels. Finally, the signals are
evaluated by fluorescence microscopy (not
shown). [From Speicher & Carter (2005) NatureRev Genet 6:782
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Chromosome-specific paints for FISH
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p p
Fluorescence in situ hybridization (FISH) – h h i i
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metaphase chromosome painting
Chromosome maintenanceO i i f li ti
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•Origins of replication•Telomeres
•Centromeres
Origins of replication
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Origins of replication
•Multiple origins-every 100 kb on average in humans
•Heterochromatin is late replicating•Replication times correspond to banding patterns•Each band replicated independently
From Miller & Therman (2001) Human Chromosomes, Springer
Telomeres
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•End structures of linear chromosomes•Serve to replicate chromosome ends•Serve to stabilize chromosome ends (i.e. prevent non-homologous end joining, NHEJ)
•G-rich tandem repeats- TTAGG, insects
- TTAGGG, vertebrates- TTTAGGG, plants
•Length is under genetic and developmental control- e.g. 2-5 kb in Arabidopsis, 60-160 kb in Tobacco, 15 kb in
humans•Sequence and proteins conserved across taxa, mammals toplants
Telomeres
FISH with a telomere-specific probe
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FISH with a telomere specific probe
Telomeres & telomerase in the replication of linear
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p
chromosome ends
Telomerase
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Telomerase
•Reverse transcriptase & RNA primer
•Repeating cycles of parental strand extension- build template for lagging strand replication- build up the number of telomeres
•Abundant in mammalian embryos, stem cells and cancer cells•Absent in mammalian somatic cells
- telomeres shorten with each cell division
- cells cease division and begin senescence
•Abundant in rapidly dividing and germ-line cells of plants•Absent in vegetative tissues of plants
Centromeres
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•Primary constriction
•Kinetochore - spindle fiber attachment•Region of sister chromatid cohesion•Constitutive heterochromatin•Repeat sequences - CENs - 5 to 170 bp
–e.g. human alphoid satellite repeat
–No universal centromere repeat, but the same repeat can befound in more than one centromere of a species or between
species
–Centromere repeats can change rapidly in evolution via
mutation, new elements, recruitment of other genomic repeats•Specific associated proteins
– e.g. Centromere-specific histone HE (CenH3)
Centromeres
A model of centromere structure
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Chromatin
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Chromatin
structure
From Miller & Therman
(2001) Human
Chromosomes, Springer
Compacts DNA ~ 10,000X
Chromatin structure –
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11 nm fiber
•Nucleosomes-147 bp DNA wound on histone core
- Histones H3, H4, H2A, H2B (2 each)
•Internucleosomal spacer-~ 60 bp linker DNA
30 nm fiber
• Histone H1 (linker) binds and compactsnucleosomes
• Exact structure is controversial
- Solenoid = single helix coiling of 11 nmfiber
- Zig-zag stacking of nucleosomes then
coiling = double helix of 11 nm fiber
From Woodcock (2006) Curr Opin Struct Biol 16:213
Chromatin structure –
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300 nm fiber
•Loops of 30 nm fibers•Attached to protein scaffold•Attachment points correspond toboundary elements, isolating
regions of differential geneexpression
Metaphase chromatin
•Coiling of the 300 nm fiber
Chromatin structure – histone modifications
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Post-translational modifications on histone proteins
• Establish global chromatin structure-heterochromatin vs euchromatin• Regulate DNA-based functions
- Transcription
- Replication, recombination & repair• Complex interactions
- Not really a simple “histone code” - “The truth is likely to be that any given modification
has the potential to activate or repress under differentconditions.”
[From Kouzarides (2007) Cell 128:693]
Chromatin structure – histone modifications
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Post-translational modifications on histone proteins alter chromatin
structure and, consequently, chromatin function
Table 1. Different Classes of Modifications Identified on Histones
Chromatin Modifications Residues Modified Functions Regulated
Acetylation K-acTranscription, Repair, Replication,
Condensation
Methylation (lysines) K-me1 K-me2 K-me3 Transcription, Repair
Methylation (arginines) R-me1 R-me2a R-me2s Transcription
Phosphorylation S-ph T-ph Transcription, Repair, Condensation
Ubiquitylation K-ub Transcription, Repair
Sumoylation K-su Transcription
ADP ribosylation E-ar Transcription
Deimination R > Cit Transcription
Proline Isomerization P-cis > P-trans Transcription
Overview of different classes of modification identified on histones. The functions that have been associated with
each modification are shown. Each modification is discussed in detail in the text under the heading of the function it
regulates. [From Kouzarides (2007) Cell 128:693]
Chromatin structure – histone modifications
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Post-translational modifications on histone proteins alter chromatin
structure and, consequently, chromatin function
Figure 1. Recruitment of Proteins to Histones (A) Domains used for the recognition of methylated
lysines, acetylated lysines, or phosphorylated serines. (B) Proteins found that associate preferentially
with modified versions of histone H3 and histone H4. [From Kouzarides (2007) Cell 128:693]
Chromatin structure – histone modifications
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Post-translational modifications on histone proteins
“The truth is likely to be that any given modification has
the potential to activate or repress under differentconditions.”
• Histone acetylation
- generally associated with activation of transcription
• Histone de-acetylation- generally associated with repression of transcription
- Histone de-acetylase targeted to methylated CpG
islands
[Kouzarides (2007) Cell 128:693]
Chromatin structure – histone modifications
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Post-translational modifications on histone proteins
“The truth is likely to be that any given modification has
the potential to activate or repress under differentconditions.”
• Lysine methyation associated with activation of
transcription: H3K4, H3K36, H3K79
• Lysine methyation associated with repression oftranscription: H3K9, H3K27, H4K20
[Kouzarides (2007) Cell 128:693]
Chromatin structure – functional consequences ofhistone modifications
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histone modifications Figure 3. Functional Consequences ofHistone Modifications (A) Gene-
expression changes are brought about by
the recruitment of the NURF complex,which contains a component BRTF
recognizing H3K4me and a component-
remodeling chromatin. (B) The Crb2
protein of fission yeast is recruited to
DNA-repair foci during a DNA-repair
response. Crb2 is partly tethered there byassociation with methylated H4 and
phosphorylated H2A. (C) The HBO1
acetyltransferase is an ING5-associated
factor and is therefore tethered to sites of
replication via methylated H3K4. HBO1
also binds to the MCM proteins found atreplication sites. Evidence exists that
HBO1 augments the formation of the
preinitiation complex and is required for
DNA replication. [From Kouzarides
(2007) Cell 128:693]
Nuclear architecture – Chromosome territories
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aAll the chromosome territories that make up the human genome can be visualized simultaneously in intact interphase
nuclei, each in a different colour. a | A red, green and blue image of the 24 labelled chromosomes (1 –
22, X and Y) wasproduced from deconvoluted mid-plane nuclear sections from a three-dimensional stack by superposition of the 7 colour
channels. b | As in 24-colour KARYOTYPING, each chromosome can be identified by using a combination labelling
scheme in which each chromosome is labelled with a different set of fluorochromes. In this way, each chromosome
territory can be automatically classified using appropriate software, which assigns the corresponding chromosome
number to a territory. If a stack of these images is collected throughout the nucleus, a simultaneous three-dimensional
reconstruction of all chromosome territories is possible. Some of the dark regions represent unstained nucleoli. For further
details see Ref. 90. | [From Speicher & Carter (2005) Nature Rev Genet 6:782]
Nuclear architecture – Chromosome territories
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•Nonrandom chromosome positioning
•Gene rich chromosomes toward center•Gene poor chromosomes toward periphery•Centromeres are not the determining factor•Chromosomes with adjacent positions more likelyto interact cytolologically
Nuclear architecture – consequences of chromosometerritories
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territories
Figure 3. Functional Consequences of Global Chromatin Organization (A and B) Spatial clustering of
genes on distinct chromosomes facilitates their expression by (A) association with shared transcription
and processing sites or (B) physical interactions with regulatory elements on separate chromosomes.
(C) The physical proximity of chromosomes contributes to the probability of chromosomal
translocations. [From Misteli (2007) Cell 128:787]
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Model of functional nuclear architectureFigure 3 Structural features that support the chromosome territory interchromatin compartment (CT IC) model are
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Figure 3. Structural features that support the chromosome-territory –interchromatin-compartment (CT –IC) model are
shown. These features are drawn roughly to scale on an optical section taken from the nucleus of a living HeLa cell.
Although experimental evidence is available to support these features, the overall model of functional nuclear
architecture is speculative (see text). a | CTs have complex folded surfaces. Inset: topological model of gene
regulation23. A giant chromatin loop with several active genes (red) expands from the CT surface into the IC space.b | CTs contain separate arm domains for the short (p) and long chromosome arms (q), and a centromeric domain
(asterisks). Inset: topological model of gene regulation78, 79. Top, actively transcribed genes (white) are located on
a chromatin loop that is remote from centromeric heterochromatin. Bottom, recruitment of the same genes (black) to
the centromeric heterochromatin leads to their silencing. c | CTs have variable chromatin density (dark brown, high
density; light yellow, low density). Loose chromatin expands into the IC, whereas the most dense chromatin is
remote from the IC. d | CT showing early-replicating chromatin domains (green) and mid-to-late-replicating
chromatin domains (red). Each domain comprises 1 Mb. Gene-poor chromatin (red), is preferentially located at the
nuclear periphery and in close contact with the nuclear lamina (yellow), as well as with infoldings of the lamina and
around the nucleolus (nu). Gene-rich chromatin (green) is located between the gene-poor compartments. e |
Higher-order chromatin structures built up from a hierarchy of chromatin fibres88. Inset: this topological view of
gene regulation27, 68 indicates that active genes (white dots) are at the surface of convoluted chromatin fibres.
Silenced genes (black dots) may be located towards the interior of the chromatin structure. f | The CT –IC model
predicts that the IC (green) contains complexes (orange dots) and larger non-chromatin domains (aggregations oforange dots) for transcription, splicing, DNA replication and repair. g | CT with 1-Mb chromatin domains (red) and IC
(green) expanding between these domains. Inset: the topological relationships between the IC, and active and
inactive genes72. The finest branches of the IC end between 100-kb chromatin domains. Top: active genes (white
dots) are located at the surface of these domains, whereas silenced genes (black dots) are located in the interior.
Bottom: alternatively, closed 100-kb chromatin domains with silenced genes are transformed into an open
configuration before transcriptional activation. [From Cremer & Cremer (2001) Nature Rev Genet 2:292]
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RIBOSOME
Just a quick overview of what we’re
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going to cover…
• What ribosome is and what its subunits are
• The purpose of ribosome
• The process of protein synthesis, including:
– DNA to mRNA (transcription)
– mRNA to protein (translation)
• Initiation
• Elongation• End of translation
Just a quick overview of what we’re
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going to cover…
• Structures of the two ribosome subunits – The larger subunit
– The smaller subunit
– RNA’s relation to their structure
What is ribosome?
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What is ribosome?
• Ribosome - protein
synthesizer consisting of
two subunits
• Larger one, “50S”, isupper picture. Smaller is
“30S”
(They look the same size
here because of spacerestrictions.)
50S and 30S???
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50S and 30S???
• Related to their respective sizes. Numbersactually measures of how quickly each subunit
sinks to the bottom of a container of liquid
when spun in a centrifuge• One subunit smaller than other, but both are
larger than average protein
A couple more nifty pictures…
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A couple more nifty pictures…
• 50S (left) and 30S. This time you can see them from
different angles, through different style of picture
So what’s the purpose of ribosome?
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So what s the purpose of ribosome?
• Ribosome basically a protein factory. Subunitseach have role in making of proteins
• To understand exactly what each subunit
does, it’s necessary to walk through proteinsynthesis step by step
Protein synthesis
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Protein synthesis
• Process starts from DNA
through “transcription”
• “Translation” is whereribosome comes in.
Translation occurs when
protein formed from code
on mRNA
•
Ribosome carries out thetranslation of the
nucleotide triplets
Protein synthesis
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Protein synthesis
• Chart - visual image of
transcription and
translation in protein
synthesizing
• DNA and RNA have
nucleotides that
determine kind of protein
•
3 nucleotides = 1 aminoacid of a protein
Ribosome and RNA
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Ribosome and RNA
• mRNA with code for proteins located at 30Ssubunit
• tRNAs responsible for carrying amino acids to
mRNA. Each tRNA has own nucleotide tripletwhich binds to matching triplet on mRNA, ex.,
tRNA with code AAA (triple adenine) would
match up with mRNA that has code UUU(triple uracil)
Initiation:
Th fi h f l i
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The first phase of translation
• Translation begins when
mRNA attaches to the 30S
• tRNA comes and binds to
mRNA where nucleotide
code matches
• This triggers 50S binding
to 30S. 50S is where all
tRNAs will bind. Now wemove on to elongation
Elongation:
Th d h
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The second phase
• Two binding sites on 50S:
A site and P site, which
aid in continuing
translation
• First tRNA connected at A
site. Now moves to P site
as another tRNA
approaches
• Second tRNA binds to A
site
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End of translation
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• Ribosome was moving
along nucleotide triplets
one by one
• Ribosome reaches “stopcodon,” peptide chainfinished. Last tRNA leaves
ribosome, leaving behind
completed peptide chain
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1st step: Initiation
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1st step: Initiation
T. Terry, U. Conn
2nd step: Elongation
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2nd step: Elongation
T. Terry, U. Conn
Last step: Termination
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Last step: Termination
T. Terry, U. Conn
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Early Evaluation of the Shape of theRibosome by EM
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Ribosome by EM
• First EM images in 1950s
• Molecules in different orientationscombined to create models
• Proteins localized by bindingantibodies
• Most early and later structuralwork on prokaryotic ribosomes
Comparative Sequence Analysis
Used to Predict Most of the
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Base Pairs
H. Noller lab web page
• First secondary structure predicted in 1980(Woese, Noller, and colleagues)
• Method first applied to tRNA (1970)and 5S rRNA (1975)
• 16S rRNA can be divided into subdomains.Much of secondary structure is local
• ~60% of nucleotides are base-paired
~1500 nt
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H. Noller lab web page
~3000 nt
Identification of Secondary Structures BySequence Analysis
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Corbino et al, Genome Biology 2005,
6:R70
• Sequence analysis can predict secondary structureby finding base-pairing potential
• When multiple related sequences are available,covariation provides additional evidence for pairing
• This figure happens to show a riboswitch (more nextTuesday on that), but the same methods were used
to deduce secondary structures of the rRNAs.
Comparative Sequence Analysis
Used to Predict Most of the
B P i
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Base Pairs
Gutell et al , Curr. Opin. Struct. Biol. (2002)
12, 301-310.
• Some tertiary interactionsshow covariation and can
therefore be predicted
•Tetraloop-receptor interactions
• Many tertiary contactsmediated by A nucleotides
• 97% of predicted base pairs werepresent in crystal structures
• 75% of base pairs in crystalstructures were predicted. Others
were not detectable because they
do not vary between sequences.
Crystal Structure of tRNA
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Fig. 19.26
• First molecular details of higher-order RNA structure, demonstrated tertiary contacts
• Two groups (A. Rich and A. Klug) published structures in 1974. First author onKlug’s work was Jon Robertus (UT Biochemistry).
High-resolution
Structure of a 16S RNP
D i
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Domain
Agalarov et al , Science (2000) 288, 107-112
• First atomic resolution view ofribosomal subdomain
• Suggests hierarchical assemblyof RNA-protein structure
70S Ribosome of Thermus
thermophilus
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Fig. 19.1
• Noller and colleagues, 5.5 Å, 2001
• Structure includes 30S, 50S, associatedproteins, and three tRNA molecules
bound in A, P, and E sites
• Showed that core and interface are
dominated by RNA, not protein
• 16S rRNA, cyan
30S proteins, blue23S rRNA, gray,
5S rRNA, dark blue
50S proteins, purple
Ribosome ‘ripped apart’to expose tRNAs
Codon-anticodon Base Pairing
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Codon anticodon Base Pairing
Fig. 19.2
• Bend in mRNA between A and P sites allows adjacent tRNAs to bind to
consecutive codons in proper orientations for peptidyl transfer
• Stereo image: Try to see the image in 3D by looking at your book
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Ribosome Schematic Based on
Structural Information
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Fig. 19.4
Structural Information
• Large cavity between subunitsto accommodate the three
tRNAs• tRNAs interact with 30S subunit
through anticodon ends and
bind to mRNA, also bound to
30S• tRNAs interact with 50S through
acceptor stems. This is where
peptidyl transfer happens
Structure of the 30S Subunit
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Fig. 19.8 Fig. 19.9
Secondary structure3D structure (same colors)
Central
domain
• Central domainstructure remains
intact
• Overall, tertiaryarrangement
dominated by RNA
50S
S
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Structure
• Monolithic RNA, not modular
• Most of protein mass onor near surface
• Portions of proteins towardmiddle are in
unprecedented, unfolded
conformations; threaded
through RNA
50S Structure Shows No Proteins NearActive Site For Peptidyl Transfer
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Active Site For Peptidyl Transfer
Fig. 19.16
Fig. 19.17
• The ribosome is a ribozyme • Proteins snake toward, but notinto, active site
Ribosome Structure and Assembly
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Ribosome Structure and Assembly
1. Introduction: The Process of Translation
2. Structures of the Ribosome and Subunits
3. Ribosome Assembly (30S)
Processing of the rRNA Precursor
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Fig. 16.4 16S 23S 5S
• E. coli has seven operons (rrn) that encode rRNAs
• Cleavage events give rise to processed rRNAs
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Binding of Some 30S Proteins Is
N F Bi di f Oth
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Necessary For Binding of Others
Fig. 19.6
• 30S reconstitution demonstrated by Nomura and colleagues (late 1960s)
[3H]-labeled S12• Added back proteins in
different orders to build up
assembly pathway
• A. S4, S7, S8, S13, S16, S20 B. S4, S8, S16, S17
C. All except S12
Sucrose gradient ultracentrifugation
The Nomura 30S Assembly Map
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• Proteins separated into primary,
secondary, and tertiary binders
• S15, S17, S4, S8, S20, S13, and S7 areprimary binders
• In general, proteins lower down on themap are on outside of 30S particle.
• Suggests similarity betweenthermodynamic pathway outlined here
and kinetic pathway in vivo
• Much less known about 50S assembly
Kinetics of 30S Reconstitution
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Traub and Nomura, J. Mol. Biol. (1969), 40, 391-413
• Very strong temperature dependence forformation kinetics
• Complete 30S subunit formation assayed byactivity in translation assay
• Proteins below dashed line in fig. 19.7 (previousslide) are not bound stably in RI intermediate
Mass Spectrometry Approach To Follow Association of
Individual Proteins With 16S rRNA
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Talkington and Williamson, Nature (2005) 438, 628-632
• Assembly initiated with *15N]-labeled proteins, then ‘chased’ with unlabeled proteins
• Extent of binding of each protein at time of chase measured by mass spectrometry
• Primary binders from Nomura map mostly bind fast
• Binding is faster in general for proteins that bind closer to 5’ end
Colors represent relative binding rates
Are Molecular Chaperones Involved in
Ribosome Assembly?
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Ribosome Assembly?
• Three of five DEAD-box proteins in E. coli are implicated in ribosome assembly
• The Hsp70 protein chaperone (DnaK) has been implicated also
• Numerous small RNAs (snoRNAs) are required, which bind transiently to regionswithin the rRNAs. These RNAs direct modifications, but some could also function
as chaperones
Key Points
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1. Prokaryotic ribosomes are composed of two subunits, the 50S and the 30S. The
50S subunit includes two rRNAs, the 23S and 5S rRNAs, and 34 proteins. The
30S subunit includes one rRNA, the 16S, and 21 proteins.
2. Recent structural analyses have revolutionized our understanding of the
ribosome. Most of the base pairs and many tertiary contacts were predictedcorrectly by comparative analysis, but the structures reveal molecular details,
interactions with proteins, and many features that were not predictable.
3. The 30S subunit can be reconstituted from pure 16S RNA and proteins. Thisprocess is thought to involve hierarchical steps of RNA folding and protein