STRUKTUR DAN FUNGSI BIOMOLEKUL

KOMPLEKS / SUPRAMOLEKUL:
MEMBRAN, KROMOSOM, RIBOSOM

Supramolecular chemistry

Supramolecular chemistry
"Supramolecular chemistry is the chemistry of the intermolecular
bond, covering the structures and functions of the entities formed
by the association of two or more chemical species"
J.-M- Lehn
"Supramolecular chemistry is defined as chemistry "beyond the
molecule", 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. Vgtle

Supramolecular chemistry in nature

Supramolecular chemistry in nature

Supramolecular chemistry in nature

Supramolecular chemistry in nature

The organic compounds from which most

cellular materials are constructed

Amino Acids

Nucleotides
Carbohydrates
Lipids

Structural hierarchy in the molecular

organization of cells

MEMBRANES

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 proteins
3. 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 a
backbone for the phospholipid
-2 fatty acids attached to the glycerol
-phosphate group attached to the glycerol

18

Phospholipids
The fatty acids are nonpolar chains of carbon
and hydrogen.
-Their nonpolar nature makes them
hydrophobic (water-fearing).
The phosphate group is polar and hydrophilic
(water-loving).

19

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 layers
together
-individual phospholipids and unanchored
proteins can move through the membrane
-saturated fatty acids make the membrane
less fluid than unsaturated fatty acids
-warm temperatures make the membrane
more fluid than cold temperatures
22

Phospholipids

23

Membrane Proteins
Membrane proteins have various functions:
1. transporters
2. enzymes
3. cell surface receptors
4. cell surface identity markers
5. cell-to-cell adhesion proteins
6. 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 b-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
Selective permeability: integral membrane
proteins allow the cell to be selective about
what passes through the membrane.
Channel proteins have a polar interior allowing
polar molecules to pass through.
Carrier proteins bind to a specific molecule to
facilitate its passage.

36

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

37

Passive Transport
Carrier proteins bind to the molecule that they
transport across the membrane.
Facilitated diffusion is movement of a molecule
from high to low concentration with the help
of a carrier protein.
-is specific
-is passive
-saturates when all carriers are occupied
38

Passive Transport
In an aqueous solution
-water is the solvent
-dissolved substances are the solutes

Osmosis is the movement of water from an area

of high to low concentration of water
-movement of water toward an area of high
solute concentration
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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 in
different ways.
1. Some cells use extrusion in which water is
ejected through contractile vacuoles.
2. Isosmotic regulation involves keeping cells
isotonic with their environment.
3. Plant cells use turgor pressure to push the
cell membrane against the cell wall and keep
the cell rigid.
43

Active Transport
Active transport
-requires energy ATP is used directly or
indirectly to fuel active transport
-moves substances from low to high
concentration
-requires the use of carrier proteins

44

Active Transport
Carrier proteins used in active transport include:
-uniporters move one molecule at a time
-symporters move two molecules in the
same direction
-antiporters move two molecules in
opposite 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 the
conformation of the carrier protein
-the affinity of the carrier protein for either
Na+ or K+ changes so the ions can be carried
across 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
Bulk transport of substances is accomplished by
1. endocytosis movement of substances
into the cell
2. exocytosis movement of materials out
of the cell

50

Bulk Transport
Endocytosis occurs when the plasma membrane
envelops food particles and liquids.
1. phagocytosis the cell takes in particulate
matter
2. pinocytosis the cell takes in only fluid
3. receptor-mediated endocytosis specific
molecules are taken in after they bind to a
receptor

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Bulk Transport
Exocytosis occurs when material is discharged
from the cell.
-vesicles in the cytoplasm fuse with the cell
membrane and release their contents to the
exterior of the cell
-used in plants to export cell wall material
-used in animals to secrete hormones,
neurotransmitters, digestive enzymes
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Modification of Cell Surfaces

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 attached
to 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 cells
connects the cytoskeletons of adjacent cells.
In tight junctions, plasma membrane proteins attach in zipper-like
fastenings; 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 the
movement 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 smooth
muscle cells to contract.

Cell-Surface Modifications:
Junctions

Extracellular Matrix
The extracellular matrix is a meshwork of polysaccharides and proteins produced by
animal cells.
Collagen gives the matrix strength and elastin gives it resilience.
Fibronectins and laminins bind to membrane receptors and permit
communication 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 the
various proteins in matrix and most likely regulate signaling proteins that bind to
receptors in the plasma protein.

CHROMOSOMES

Chromosomes:
Chromosome structure
Chromatin structure
Chromosome variations
The new cytogenetics

Prokaryotic chromosomes
Circular double helix
Complexed with protein in a
structure termed the nucleoid
Attached to plasma
membrane

Eukaryotic Chromosomes
Located in the nucleus
Each chromosome consists of a single molecule of
DNA and its associated proteins
The DNA and protein complex found in eukaryotic
chromosomes is called chromatin
1/3 DNA and 2/3 protein
Complex interactions between proteins and nucleic
acids in the chromosomes regulate gene and
chromosomal function

Some evidence that chromosomes contain a

single DNA molecule
Pulsed-field gel electrophoresis - separation of
chromosomes
Analysis of the complete nucleotide sequence
of many genomes now
In situ hybridization (below)

Karyotype
The representation of entire metaphase chromosomes in a
cell, arranged in order of size and other characteristics

Ideogram
Diagramatic representation
of a karyotype
Individual chromsomes are
recognized by
-arm lengths
p, short
q, long
-centromere position
metacentric
sub-metacentric
acrocentric
telocentric

-staining (banding) patterns

From Miller & Therman (2001) Human
Chromosomes, Springer

Chromsome banding
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

C-banded karyotype of XY cell

From Miller & Therman (2001) Human

Chromosomes, Springer

Constitutive heterochromatin
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

Facultative heterochromatin

All types of sequences

C-banding negative
Late replicating
Condensed conformation
Not transcribed
Includes genes silenced in specific cell types
and/or at specific times in development
e.g. inactivated X chromosomes

Euchromatin
Actively expressed sequences
More open conformation

Fluorescence in situ
hybridization (FISH)

a | The basic elements of fluorescence in situ

hybridization are a DNA probe and a target
sequence. b | Before hybridization, the DNA probe
is labelled by various means such as NICK
TRANSLATION, 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) Nature
Rev Genet 6:782]

Fluorescence in situ hybridization (FISH)

[From Speicher & Carter (2005) Nature Rev Genet 6:782]

a | Painting probes stain entire chromosomes. b | Regional painting probes can be generated by chromosome
microdissection c | Centromeric-repeat probes are available for almost all human chromosomes. d | Large-insert clones
are available for most genomic regions. Subtelomeric probes, which are often used to screen for cryptic translocations
that are not usually visible in conventional chromosome-banding analyses, are shown in this example. e | Special probe
sets can be designed to facilitate diagnosis of known structural rearrangements. In this example, the probe set includes
a breakpoint-spanning probe (red) and two breakpoint-flanking probes (green and blue). f | Genomic DNA is used as the
probe in comparative genomic hybridization (CGH) to establish copy number. An analysis of chromosome 8 is shown as
an example. Simultaneous visualization of both test DNA (green region) and normal reference DNA (red region)
fluorochromes shows balanced regions in orange (equal amounts of green and red fluorochromes). g | For highresolution analysis, DNA fibres can be used as the target for probe hybridization. The simultaneous hybridization of two
different probes is shown, labelled green and red. h | Microarrays can be used as targets for hybridization to provide
resolutions down to the single-nucleotide level. A BAC array is shown, to which test DNA and reference DNA are
hybridized. Individual clones show different colours after hybridization depending on whether the corresponding DNA in
the test sample is lost (red on the array), gained (green on the array) or neither (yellow on the array).

Fluorescence in situ hybridization (FISH) probes on

metaphase chromosomes

Chromosome-specific paints for FISH

Fluorescence in situ hybridization (FISH)

metaphase chromosome painting

Chromosome maintenance
Origins of replication
Telomeres
Centromeres

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
End structures of linear chromosomes
Serve to replicate chromosome ends
Serve to stabilize chromosome ends (i.e. prevent nonhomologous 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 to
plants

FISH with a telomere-specific probe

Telomeres & telomerase in the replication of linear

chromosome ends

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
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 be
found 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)

A model of centromere structure

Chromatin
structure
Compacts DNA ~ 10,000
X

From Miller & Therman

(2001) Human
Chromosomes, Springer

Chromatin structure
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 compacts
nucleosomes
Exact structure is controversial
- Solenoid = single helix coiling of 11 nm
fiber
- Zig-zag stacking of nucleosomes then
coiling = double helix of 11 nm fiber
From Woodcock (2006) Curr Opin Struct Biol 16:213

Chromatin structure
300 nm fiber
Loops of 30 nm fibers
Attached to protein scaffold
Attachment points correspond to
boundary elements, isolating
regions of differential gene
expression

Metaphase chromatin
Coiling of the 300 nm fiber

Chromatin structure histone modifications

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 different
conditions.
[From Kouzarides (2007) Cell 128:693]

Chromatin structure histone modifications

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-ac

Transcription, 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

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

Post-translational modifications on histone proteins
The truth is likely to be that any given modification has
the potential to activate or repress under different
conditions. [Kouzarides (2007) Cell 128:693]
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

Chromatin structure histone modifications

Post-translational modifications on histone proteins
The truth is likely to be that any given modification has
the potential to activate or repress under different
conditions. [Kouzarides (2007) Cell 128:693]
Lysine methyation associated with activation of
transcription: H3K4, H3K36, H3K79
Lysine methyation associated with repression of
transcription: H3K9, H3K27, H4K20

Chromatin structure functional consequences of

histone modifications
Figure 3. Functional Consequences of
Histone Modifications (A) Geneexpression changes are brought about by
the recruitment of the NURF complex,
which contains a component BRTF
recognizing H3K4me and a componentremodeling chromatin. (B) The Crb2
protein of fission yeast is recruited to
DNA-repair foci during a DNA-repair
response. Crb2 is partly tethered there by
association 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 at
replication 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

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 (122, X and Y) was
produced 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

Nonrandom chromosome positioning
Gene rich chromosomes toward center
Gene poor chromosomes toward periphery
Centromeres are not the determining factor
Chromosomes with adjacent positions more likely
to interact cytolologically

Nuclear architecture consequences of chromosome

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]

Nuclear architecture Nuclear factories

Figure 1. Compartmentalization
of Nuclear Processes
Transcription, replication, and
DNA repair are
compartmentalized. (A)
Transcription sites visualized
by incorporation of bromo-UTP,
(B) replication sites visualized
by incorporation of bromodUTP, and (C) repair sites
visualized by accumulation of
repair factor 53BP1 at a
double-strand break (DSB) are
shown. In all cases,
components are dynamically
recruited from the nucleoplasm
as single subunits or small
preassembled subcomplexes.
(A) is reprinted with permission from Elbi et al., 2002, (B) is courtesy of Rong Wu and David Gilbert at
Florida State University, and (C) is courtesy of Evi Soutoglou from the National Cancer Institute, NIH.
[From Misteli (2007) Cell 128:787]

Model of functional nuclear architecture

Figure 3. Structural features that support the chromosome-territoryinterchromatin-compartment (CTIC) 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). [From Cremer & Cremer (2001) Nature Rev Genet 2:292]

Model of functional nuclear architecture

Figure 3. Structural features that support the chromosome-territoryinterchromatin-compartment (CTIC) 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 CTIC model
predicts that the IC (green) contains complexes (orange dots) and larger non-chromatin domains (aggregations of
orange 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]

RIBOSOME

Just a quick overview of what were

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 were

going to cover
Structures of the two ribosome subunits
The larger subunit
The smaller subunit
RNAs relation to their structure

What is ribosome?
Ribosome - protein
synthesizer consisting of
two subunits
Larger one, 50S, is
upper picture. Smaller is
30S
(They look the same size
here because of space
restrictions.)

50S and 30S???

Related to their respective sizes. Numbers
actually 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

50S (left) and 30S. This time you can see them from
different angles, through different style of picture

So whats the purpose of ribosome?

Ribosome basically a protein factory. Subunits
each have role in making of proteins
To understand exactly what each subunit
does, its necessary to walk through protein
synthesis step by step

Protein synthesis
Process starts from DNA
through transcription
Translation is where
ribosome comes in.
Translation occurs when
protein formed from code
on mRNA
Ribosome carries out the
translation of the
nucleotide triplets

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 amino
acid of a protein

Ribosome and RNA

mRNA with code for proteins located at 30S
subunit
tRNAs responsible for carrying amino acids to
mRNA. Each tRNA has own nucleotide triplet
which 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:
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 we
move on to elongation

Elongation:
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

Elongation (continued)
Peptide bond forms
between amino acids of
tRNAs (methionine and
proline)
First tRNA now detached
from its amino acid, and it
leaves ribosome. Second
tRNA still has proline and
methionine attached

Elongation (continued)
The tRNA left now moves
to P site. Ribosome ready
to accept another tRNA
and continue process
Each tRNA adds another
amino acid to growing
peptide chain (thus
elongation)
Eventually process has to
finish, however

End of translation
Ribosome was moving
along nucleotide triplets
one by one
Ribosome reaches stop
codon, peptide chain
finished. Last tRNA leaves
ribosome, leaving behind
completed peptide chain

End of translation (continued)

Ribosome separates from
mRNA
Ribosome subunits also
separate, and will remain
this way until another
mRNA comes along to
restart the process

1st step: Initiation

T. Terry, U. Conn

2nd step: Elongation

T. Terry, U. Conn

Last step: Termination

T. Terry, U. Conn

Ribosome Structure and Assembly

1. Introduction: The Process of Translation

2. Structures of the Ribosome and Subunits

- Predictions from sequence analysis
- x-ray crystallography determinations
3. Ribosome Assembly (30S)

Early Evaluation of the Shape of the

Ribosome by EM
First EM images in 1950s
Molecules in different orientations
combined to create models
Proteins localized by binding
antibodies

Most early and later structural

work on prokaryotic ribosomes

Comparative Sequence Analysis

Used to Predict Most of the
Base Pairs

Method first applied to tRNA (1970)

and 5S rRNA (1975)
First secondary structure predicted in 1980
(Woese, Noller, and colleagues)
16S rRNA can be divided into subdomains.
Much of secondary structure is local
~60% of nucleotides are base-paired

~1500 nt
H. Noller lab web page

~3000 nt
H. Noller lab web page

Identification of Secondary Structures By

Sequence Analysis

Sequence analysis can predict secondary structure

by finding base-pairing potential
When multiple related sequences are available,
covariation provides additional evidence for pairing
This figure happens to show a riboswitch (more next
Tuesday on that), but the same methods were used
to deduce secondary structures of the rRNAs.
Corbino et al, Genome Biology 2005, 6:R70

Comparative Sequence Analysis

Used to Predict Most of the
Base Pairs
97% of predicted base pairs were
present in crystal structures
75% of base pairs in crystal
structures were predicted. Others
were not detectable because they
do not vary between sequences.
Some tertiary interactions
show covariation and can
therefore be predicted
Tetraloop-receptor interactions
Many tertiary contacts
mediated by A nucleotides
Gutell et al, Curr. Opin. Struct. Biol. (2002)
12, 301-310.

Crystal Structure of tRNA

First molecular details of higher-order RNA structure, demonstrated tertiary contacts
Two groups (A. Rich and A. Klug) published structures in 1974. First author on
Klugs work was Jon Robertus (UT Biochemistry).

Fig. 19.26

High-resolution
Structure of a 16S RNP
Domain
First atomic resolution view of
ribosomal subdomain
Suggests hierarchical assembly
of RNA-protein structure

Agalarov et al, Science (2000) 288, 107-112

70S Ribosome of Thermus

thermophilus
Noller and colleagues, 5.5 , 2001
Structure includes 30S, 50S, associated
proteins, and three tRNA molecules
bound in A, P, and E sites
16S rRNA, cyan
30S proteins, blue
23S rRNA, gray,
5S rRNA, dark blue
50S proteins, purple
Showed that core and interface are
dominated by RNA, not protein
Ribosome ripped apart
to expose tRNAs
Fig. 19.1

Codon-anticodon Base Pairing

Bend in mRNA between A and P sites allows adjacent tRNAs to bind to
consecutive codons in proper orientations for peptidyl transfer

Fig. 19.2

Stereo image: Try to see the image in 3D by looking at your book

Bridges Between the 30S and 50S Subunits

16S and 23S rRNA, gray
5S rRNA, blue
Proteins, light blue
RNA-RNA bridges, pink
Protein-protein bridges, yellow

Fig. 19.3

Most of the intersubunit bridges are RNA

All bridges near tRNAs are RNA
Translocation requires large movements:
some bridges may be dynamic

Ribosome Schematic Based on

Structural Information
Large cavity between subunits
to 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

Fig. 19.4

Structure of the 30S Subunit

3D structure (same colors)

Secondary structure

Central
domain

Central domain
structure remains
intact

Fig. 19.8

Overall, tertiary
arrangement
dominated by RNA

Fig. 19.9

50S
Structure
Monolithic RNA, not modular
Most of protein mass on
or near surface
Portions of proteins toward
middle are in
unprecedented, unfolded
conformations; threaded
through RNA

50S Structure Shows No Proteins Near

Active Site For Peptidyl Transfer
The ribosome is a ribozyme

Proteins snake toward, but not

into, active site

Fig. 19.17
Fig. 19.16

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

E. coli has seven operons (rrn) that encode rRNAs
Cleavage events give rise to processed rRNAs

Fig. 16.4

16S

23S

5S

2D Gel Electrophoresis Shows

Protein Components
Method first used to ribosomal proteins in 1970
Ribosomes isolated, then proteins separated on gel
Here, two dimensions were native PAGE at two
different pH values and acrylamide concentrations

30S: 21 proteins

Fig. 19.5

Eukaryotic ribosome is more complex:

- 40S (30S equivalent) has 30 proteins
- 60S (50S equivalent) has 40 proteins and 3
RNAs (28S, 5.8S, and 5S)

50S: 34 proteins

Binding of Some 30S Proteins Is

Necessary For Binding of Others
30S reconstitution demonstrated by Nomura and colleagues (late 1960s)
Added back proteins in
different orders to build up
assembly pathway

[3H]-labeled S12

A. S4, S7, S8, S13, S16, S20

B. S4, S8, S16, S17
C. All except S12

Sucrose gradient ultracentrifugation

Fig. 19.6

The Nomura 30S Assembly Map

Proteins separated into primary,
secondary, and tertiary binders
S15, S17, S4, S8, S20, S13, and S7 are
primary binders
In general, proteins lower down on the
map are on outside of 30S particle.
Suggests similarity between
thermodynamic pathway outlined here
and kinetic pathway in vivo

Much less known about 50S assembly

Kinetics of 30S Reconstitution

Complete 30S subunit formation assayed by
activity in translation assay

Very strong temperature dependence for

formation kinetics

Proteins below dashed line in fig. 19.7 (previous

slide) are not bound stably in RI intermediate

Traub and Nomura, J. Mol. Biol. (1969), 40, 391-413

Mass Spectrometry Approach To Follow Association of

Individual Proteins With 16S rRNA
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

Talkington and Williamson, Nature (2005) 438, 628-632

Are Molecular Chaperones Involved in

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 regions
within the rRNAs. These RNAs direct modifications, but some could also function
as chaperones

Key Points
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 predicted
correctly 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. This
process is thought to involve hierarchical steps of RNA folding and protein
binding, but many features remain poorly understood.