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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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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


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


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]



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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]



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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]



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“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]



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“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