Transcript Bacterial Genetics

Bacterial Genetics
Classification and Taxonomy
Phylogeny
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The most recent model for the basic divisions of life is
the “three domain model”, first put forth by Carl Woese
in the 1970’s.
He compared the sequences of 16S ribosomal RNA
genes, which are fundamentally important for protein
synthesis and found in all known living organisms.
He discovered that “bacteria” could be divided into 2
very different groups, the Eubacteria (often just called
Bacteria) and the Archaea
The third group is the eukaryotes, organisms in which
the DNA is contained within a membrane-bound
nucleus.
Eubacteria and Archaea are the two type of prokaryote,
organisms in which the DNA is loose within the
cytoplasm and not contained within a nucleus.
Archaea usually live in extreme environments: very
hot, acidic, salty, etc. They use quite different
information processing machinery than the bacteria.
We are going to mostly ignore them.
Classifying Bacteria
• Classically, bacteria have been characterized by
their staining pattern, shape, reaction to oxygen,
pH, temperature, and salt optima, and their ability
to metabolize various compounds.
– good functional classification: what they look like, and
where they live, but often evolutionary relationships are
not accurate
• More recent classification schemes are based in
16S ribosomal RNA., which is found in all
(known) cells. Also, the percentage of G and C
(G+C content) is used for classification.
Gram Stain
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A major distinction between groups
of bacteria is based on the Gram
stain. In this method, bacteria are
treated with the dye “crystal
violet”, then washed. Often a
second stain, “safranin” is applies
to make the unstained bacteria
visible.
Gram stain causes bacteria with a
lot of peptidoglycan and very little
lipid in their cells walls to stain
purple. The presence or absence of
peptidoglycan is a fundamental
biochemical difference between
groups of bacteria
Another stain, the “acid-fast stain”
is used to identify Mycobacteria,
such as the tuberculosis agent
Mycobacterium tuberculosis.
Bacterial Morphology
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Bacteria only take a few basic shapes, which are
found in many different groups. Bacterial cells
don’t have internal cytoskeletons, so their shapes
can’t be very elaborate.
Shape: coccus (spheres) and bacillus (rods).
Spirillum (spiral) is less common.
– note: “bacillus” is a shape, but “Bacillus” or
(better) Bacillus is a taxonomic group, a
genus containing such species as Bacillus
subtilis, Bacillus anthracis, and Bacillus
megaterium. The bacillus shape is NOT
limited to the Bacillus genus.
Aggregation of cells: single cells, pairs (diplo),
chains (strepto), clusters (staphylo).
Thus we have types such as diplococcus (pair of
spheres) and streptobacillus (chain of rods).
Relationship to Oxygen
• For more than half of Earth’s history, oxygen
wasn’t present in the atmosphere. Many bacteria
evolved under anaerobic conditions.
• Classification:
– strict aerobes (need oxygen to survive)
– microaerobes need oxygen, but at reduced
concentration (such as in cow guts)
– strict anaerobes (killed by oxygen)
– aerotolerant (don’t use oxygen, but survive it).
– facultative anaerobes (use oxygen when it is present,
but live anaerobically when oxygen is absent).
Temperature
• thermophiles have an optimum growth
temperature above 50oC
• hyperthermophiles have an optimum growth
temperature above 80oC. Many of these are
Archaea, not Bacteria
• psychrophiles (cryophiles) have an optimum
growth temperature below 15oC
• mesophiles are those with optima between 15oC
and 50oC.
Metabolic Classification
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All living organisms need to obtain energy from the environment, and they need to
obtain or make reduced, organic carbon compounds.
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CO2 (carbon dioxide) is the most oxidized form of carbon, and it is not considered “organic”
Energy comes from 2 sources, sunlight or chemical bonds
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an organism that uses light for energy is a phototroph
an organism that uses chemical bonds for energy is a chemotroph
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chemotrophs are sub-divided:
• if the chemical bonds used for energy come from organic molecules, it is a
chemoorganotroph.
• If inorganic compounds are used, it is a chemolithotroph (litho = rock)
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Organic carbon compounds are often obtained from other organisms: heterotroph.
Or, organic compounds can be made by reducing carbon dioxide: autotroph.
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Humans are thus chemoorganoheterotrophs. Plants are photoautotrophs. Various
bacteria are found in all 6 roles.
Tree of Life: Bacterial Phyla
http://tolweb.org/tree?group=Eubacteria&contgroup=Life_on_Earth
Bacterial Structure
Structure of Bacteria
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All cells have 3 main components:
– DNA (‘nucleoid”)
• genetic instructions
– surrounding membrane (“cytoplasmic
membrane”)
• limits access to the cell’s interior
– cytoplasm, between the DNA and the
membrane
• where all metabolic reactions occur
• especially protein synthesis, which occurs on
the ribosomes
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Bacteria also often have these features:
– cell wall
• resists osmotic pressure
– flagella
• movement
– pili
• attachment
– capsule
• protection and biofilms
Cell Envelope
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The cell envelope is all the layers
from the cell membrane outward,
including the cell wall, the
periplasmic space, the outer
membrane, and the capsule.
– All free-living bacteria have a
cell wall
– periplasmic space and outer
membrane are found in Gramnegatives
– the capsule is only found in some
strains
Cell Membrane
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The cell membrane (often called the plasma
membrane) is composed of 2 layers of
phospholipids.
Phospholipids have polar heads and non-polar
tails.
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“Polar” implies that the heads are hydrophilic:
they like to stay in an aqueous environment:
facing the outside world and the inside of the
cell.
“non-polar” means that the tails are
hydrophobic: they want to be away from water,
in an oily environment. The tails are in the
center of the membrane
A pure phospholipid membrane only allows
water, gasses, and a few small molecules to
move freely through it.
Membrane Proteins
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Proteins float in the membrane like ships
on the surface of the sea: the fluid-mosaic
model.
Peripheral membrane proteins are bound
to one surface of the membrane.
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Some attached to the cell membrane by a
fatty acid covalently attached to one of the
protein’s amino acids
Others are attached by stretches of
hydrophobic amino acids of the protein’s
surface
Integral membrane proteins are
embedded in the membrane by one or
more stretches of hydrophobic amino
acids. Many of these proteins transport
molecules in and out of the cell. The
transport proteins are very selective: each
type of molecule needs its own
transporter.
Transport Across the Cell Membrane
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Basic rule: things spontaneously move from
high concentration to low concentration
(downhill). This process is called diffusion.
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Getting many molecules into the cell is simply a
matter of opening up a protein channel of the
proper size and shape. The molecules then move
into the cell by diffusing down the concentration
gradient. Passive transport, or facilitated
diffusion.
To get things to move from low to high (uphill),
you need to add energy: the molecules must be
pumped into the cell. Pumps are driven by ATP
energy. Active transport.
More Membrane Transport
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Often2 molecules are transported
together, with one moving by diffusion
down its concentration gradient and the
other carried along up its concentration
gradient.
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If the two molecules move in the same
direction, the protein channel is a
symporter. See the diagram of the
sodium-glucose symport mechanism
If the two molecules move in opposite
directions, the channel is an antiporter.
Cell Wall
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Osmotic pressure is the force generated by
water attempting to move into the cell.
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Water can go through the cell membrane freely
The contents of the cell are very concentrated
Like all things, water moves from areas of high
concentration to areas of low concentration. This
means, water will move from outside the cell
(dilute environment) to inside (concentrated
environment).
Osmotic pressure can easily cause a cell to swell
up and burst.
Bacteria, along with plants and fungi, resist
osmotic pressure by surrounding the cell in a
rigid box, the cell wall.
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Composed of peptidoglycan (also called
proteoglycan or murein)
Long chains of polysaccharide cross-linked by
short peptides (amino acid chains).
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The peptides contain the unusual mirror-image
amino acids D-alanine and D-glutamate
polysaccharide is composed of alternating “amino
sugars”: N-acetylglucosamine and Nacetylmuramic acid
More Cell Wall
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Gram-positive vs Gram-negative are defined by the
structure of the cell wall
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the Gram stain binds to peptidoglycan
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Gram-positive: many layers of peptidoglycan, which
is anchored to the cell membrane by teichoic acid.
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Gram-negative: 1-2 layers of peptidoglycan = thin
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The periplasmic space is between the cell membrane
and the cell wall. It contains enzymes and other
proteins, such as chemoreceptors for sensing the
environment.
Outside the peptidglycan layer is the “outer membrane”.
It is pierced by porins: protein channels, and its out
surface is covered with lipopolysaccharides (sugars
linked to membrane lipids), which are often antigenic
and or toxic.
Capsule
Some bacteria (often
pathogens) are surrounded by a
thick polysaccharide capsule.
This is a loose jelly-like or
mucus-like layer. It helps
prevent immune system cells
from reaching the bacteria, and
it forms part of biofilms.
Membrane Structures
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Pili (singular = pilus) are hairs projecting from the
surface. They are composed of pilin protein. There are
several types:
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DNA can be transferred between bacteria by conjugation,
which is initiated when sex pili on the donor cell attach to
and draw in the recipient cell.
Fimbriae (singular = fimbria) are pili used to attach the
bacteria to target cells ( in infection) or to surfaces, where
they form a biofilm.
Flagella are long hairs used to propel the cells. They are
composed of flagellin protein.
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At the base of each flagellum is a motor embedded in the
membrane and cell wall. It turns in a rotary motion, driven
by proton-motive force (the flow of protons i.e. H+ ions
across the cell membrane).
The suffix “-trichous” is used to describe the placement of
flagella: e.g. lophotrichous = several flagella all clustered
at one end.
Chemotaxis
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The flagellar motor is reversible:
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Counterclockwise rotation: bacterium
moves in a straight line
– clockwise rotation: bacterium tumbles
randomly
– the motor periodically reverses, causing
a random change in direction: bacteria
move in a random walk.
chemotaxis: bacteria move toward the source
of nutrients by swimming up the chemical
gradient. Or, away from a toxin.
– When moving up the gradient, bacteria
rarely tumble, but when moving across
it, or in the opposite direction, tumbling
is frequent.
– This produces a net motion in the
proper direction
Spores
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Some bacteria can form very tough spores, which are
metabolically inactive and can survive a long time
under very harsh conditions.
– Allegedly, some bacterial spores that were embedded in
amber or salt deposits for 25 million years have been
revived. These experiments are viewed skeptically by
many scientists.
– Panspermia: the idea that life got started on Earth due to
bacterial spores that drifted in from another solar
system. (However, it still had to start somewhere!).
• “Extraordinary claims demand extraordinary proof”
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Spores can also survive very high or low temperatures
and high UV radiation for extended periods. This
makes them difficult to kill during sterilization.
– Anthrax
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Spores are produced only by a few genera in the
Firmicutes:
– Bacillus species including anthracis (anthrax) and
cereus (endotoxin causes ~5% of food poisoning)
– Clostridium species including tetani (tetanus),
perfringens (gangrene), and botulinum (botulism: food
poisoning from improperly canned food)
Metabolism
Oxidation-Reduction
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Two truisms:
– Chemistry is the study of the movement of
electrons between atoms
– Life is applied chemistry
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Oxidation: a molecule loses an electron
– LEO: Lose Electron Oxidation
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Reduction: a molecule gains an electron
– GER: Gain Electron Reduction
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In living cells, there are no free electrons: every
time an electron leaves one molecule, it goes to
another one.
– Thus, all oxidation reactions are coupled with
reduction reactions: one compound is oxidized
while the other is reduced. “Redox” reactions
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Electrons are often accompanied by H+ ions.
Thus, FAD is the oxidized form, and FADH2 is
the reduced form: it has 2 more electrons (and
H’s) than FAD.
– For this reason, enzymes that perform oxidations
are usually called dehydrogenases.
Redox Potential
• Redox potential is a measure of the affinity of compounds for
electrons. The more positive a compound’s redox potential is, the
greater its tendency to acquire electrons.
– Redox potential is measured in millivolts (mV), relative to hydrogen at 1
atm pressure. Compounds are at 1 M concentration.
• H2  2 H+ + 2 e• The idea is, if your compound was mixed with hydrogen gas, would electrons
flow from your compound to the hydrogen (compound has a negative redox
potenitial), or from the hydrogen to your compound (compound has a positive
redox potenital)?
– Redox potential is affected by the concentration of the reactants and also
by the redox potential of the environment
• When electrons in compounds with lower (more negative) redox
potentials are moved to compounds with higher potentials, energy is
released. Organisms capture this energy to live on.
Some Redox Reactions
Used in Bacteria
Some Lithotrophic Reactions
Fermentations
•Fermentation is defined as a process where organic
molecules are both the electron
donor and the electron acceptor.
•Since the complete oxidation of organic molecules
ends at carbon dioxide, fermentations are by
definition incomplete oxidations: there is always
some potential energy left in the products of a
fermentation.
•And thus the fermentation products excreted
by one species are often used as food sources
for another species.
•The best known fermentations involve the products
of glycolysis.
•Glucose is oxidized to pyruvate, but the electrons
from glucose are used to convert NAD+ to NADH.
•To get rid of these electrons, NADH is used to
reduce pyruvate to lactate (as in anaerobic human
muscle) or to ethanol (as in yeast). Both of these
pathways are used in various bacteria.
Other Fermentations
Aerobic Respiration
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The most efficient way of
producing energy is by oxidizing
organic compounds to carbon
dioxide = respiration. This is the
process used by most eukaryotes
and aerobic bacteria.
Energy Generation
• Energy in the cell is generated
and used in the form of ATP.
• Two basic way s of generating
ATP:
– substrate-level
phosphorylation. The simplest
form: transfering a phosphate
group from another molecule
to ADP, creating ATP.
– chemiosmotic: generation of a
proton (H+) gradient across a
membrane. This gradient is
called “proton-motive force”.
Chemiosmotic Theory
•The same basic process in the mitochondria
as in many bacteria.
•High energy electrons from an electron
donor are used to pump H+ ions out of the
cell, into the periplasmic space
•This drains energy from the electrons
•Electron transport
•There are thus more H+ ions outside than
inside: the pH outside is lower than inside.
•The H+ ions are then allowed back into the
cell by passing them through the ATP
synthase protein, which uses the energy of
the H+ ions flowing down the gradient to
attach phosphate (Pi) to ADP, creating ATP.
•the gradient is both chemical: more H+
outside than inside, and electrical: more +
charge outside than inside
Carbon Assimilation
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Heterotrophic organisms obtain
organic carbon compounds from
pre-existing organic molecules.
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often the same molecules they are
using for energy: glucose for
example.
Autotrophs “fix” carbon dioxide into
organic carbon.
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4 pathways:
Calvin cycle (ribulose bis-phosphate
pathway. Used in plants and many
bacteria: the most common pathway
Reductive TCA cycle: run the Krebs
cycle backwards
Reductive acetyl CoA pathway,
which requires hydrogen gas and
produces carbon monoxide as an
intermediate.
3-hydroxypropiuonate cycle. Seems
to mostly be in Archaea
Carbon Assimilation Pathways
Nitrogen Assimilation
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Most of the nitrogen on Earth is
nitrogen gas, N2, which is strongly held
together by a triple bond.
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Nitrogen’s major use in the cell is as a
component of amino acids, in the
ammonium form: -NH2
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nitrogen fixation, converting nitrogen to
ammonia, is very energy-intensive and
carried out by a small group of bacteria,
including some Clostridium.
some nitrogen is also fixed by lightning.
Many organisms get their nitrogen from
organic nitrogen compounds
some organisms perform
ammonification, which means splitting
the amino group off organic
compounds, releasing ammonium ions.
Nitrogen is also found as nitrate. Most
bacteria can reduce nitrate (NO3-) to
nitrite (NO2-) and then to ammonia:
assimilatory nitrate reduction.
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nitrate is also degraded back into
nitrogen gas by other bacteria:
denitrification.
the reverse process, converting
ammonia into nitrate and nitrite, is used
as an energy source by some
lithotrophs. It is called nitrification.
More Nitrogen Assimilation
• When nitrogen is taken into the
cell in the form of ammonium
ions, it is attached to glutamate,
forming glutamine, using the
enzyme glutamine synthetase.
• Alpha-ketoglutarate, glutamate,
and glutamine can all be
interconverted.
– this is the source of the amino group
of amino acids and amino sugars.
Assimilation of Other Elements
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Cells use several other elements: C,
H, O, and N are the major ones
– also covalently bound: P, S, Se
– ions: Na, K, Mg, Ca, Cl
– trace elements (mostly as enzyme
co-factors): Fe, Mn, Co, Cu, Ni,
Zn, others....
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Sulfur can be incorporated from
organic sources, but it is often
taken into the cell as sulfate (SO42). Getting into the cell requires
attaching it to the ATP derivative
APS, after which it is reduced to
sulfide (S-2) and then attached to
serine, converting it to cysteine.
phosphate (PO4-3) is generally
found in the same form as it is
used. It just needs to be
transported into the cell.
Intermediary Metabolism
• Lots of interconversions.
• it is necessary to make:
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amino acids (proteins)
nucleotides (DNA, RNA, and ATP)
sugars (part of nucleotides, food, structure)
lipids: food storage and membrane
several co-factors for enzymes: biotin, cytochromes, panthothenic
acid, NAD, riboflavin, cobalamin, ubiquinone, etc.
• The central metabolic pathways of glycolysis and the
Krebs cycle have several side branches that feed these
biosynthetic pathways.
Enzyme Nomenclature
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Every chemical interconversion requires an enzyme to catalyze it.
Nearly all enzyme names end in –ase
Enzyme functions: which reactants are converted to which products
– Across many species, the enzymes that perform a specific function are usually
evolutionarily related. However, this isn’t necessarily true. There are cases of two entirely
different enzymes evolving similar functions.
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Enzyme functions are given unique numbers by the Enzyme Commission.
– E.C. numbers are four integers separated by dots. The left-most number is the least
specific
– For example, the tripeptide aminopeptidases have the code "EC 3.4.11.4", whose
components indicate the following groups of enzymes:
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EC 3 enzymes are hydrolases (enzymes that use water to break up some other molecule)
EC 3.4 are hydrolases that act on peptide bonds
EC 3.4.11 are those hydrolases that cleave off the amino-terminal amino acid from a polypeptide
EC 3.4.11.4 are those that cleave off the amino-terminal end from a tripeptide
Top level E.C. numbers:
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E.C. 1: oxidoreductases (often dehydrogenases): electron transfer
E.C. 2: transferases: transfer of functional groups (e.g. phosphate) between molecules.
E.C. 3: hydrolases: splitting a molecule by adding water to a bond.
E.C. 4: lyases: non-hydrolytic addition or removal of groups from a molecule
E.C. 5: isomerases: rearrangements of atoms within a molecule
E.C. 6: ligases: joining two molecules using energy from ATP
Detailed Pathways
• For many compounds, there can be more than one way to
produce it. Some organisms have more than one pathway
to a given compound, and sometimes different organisms
produce it by different mechanisms.
• KEGG (Kyoto Encyclopedia of Genes and Genomes) has a
comprehensive set of pathway maps, with individual
species differences noted.
– KEGG also has information about individual enzymes and ligands.
You can get there by clicking the elements of the map.
– http://www.genome.ad.jp/kegg/pathway.html
Genes and Gene Expression
Polypeptides and Proteins
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All of the work in the cell: energy generation, synthesis of
new components, response to environmental stimuli, etc., is
performed by proteins.
Proteins are primarily composed of polypeptides: linear
chains of amino acids.
– each polypeptide is called a “subunit”.
• each gene produces one type of polypeptide
– some proteins are composed of a single polypeptide, while
others have 2 or more (up to maybe 20 in very complex
proteins) subunits
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Proteins, especially enzymes, also often have co-factors
bound to them.
– some co-factors are just metal ions such as Zn+2
– others are more complex: most of the human “vitamins” are
enzyme co-factors
• in many species of bacteria, co-factors are synthesized by complex
pathways.
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Some proteins have sugars, lipids, or other small molecules
attached to them.
Gene Expression
• Most genes code for
polypeptides
– maybe 5% of genes produce
special RNA molecules only
• The general process of gene
expression is to transcribe an
RNA copy of the gene, called
messenger RNA (mRNA). The
mRNA is then translated into
the polypeptide by the action of
ribosomes.
– this is sometimes referred to as
the Central Dogma of
Molecular Biology
Genes on the Chromosome
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The bacterial chromosome is a very long
molecule of DNA.
– The genes are short regions of this molecule.
– A typical bacterial genome has 2000-5000
genes.
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the position of each gene on the
chromosome is the same in all members of a
species
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lines
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there is some local clustering of genes in the
same biochemical pathway, but in general
position on the chromosome cannot be
correlated with gene function.
Genes have a particular orientation on the
DNA strand: they are written with the 5’ end
on the left and the 3’ end on the right.
– However, either strand of the DNA can
encode a gene. The result of this is that
genes on one strand face in one direction,
and genes one the other strand face the other
direction
Transcription
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Gene expression begins when the enzyme RNA
polymerase binds to the promoter region just upstream
from the gene.
– the promoter consists of 2 segments of important
nucleotides, with spacers (whose sequence doesn’t matter) in
between.
– positioned -10 and -35 bp upstream from RNA start
– it’s a consensus sequence: variations on a common theme.
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Transcription is said to start at the 5’ end of the mRNA and
end at the 3’ end. This refers to the free ends of the ribose
sugar in the RNA molecule.
The RNA polymerase then moves down the DNA, using
one DNA strand as a template to synthesize an RNA copy
of the gene.
– the raw materials are “NTPs”: nucleoside triphosphates. The
energy needed to do the synthesis come from removing the 2
terminal phosphate groups, the same process as in using ATP
energy.
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Transcription ends at a terminator sequence. The RNA
polymerase falls off the DNA, releasing the new mRNA.
In eukaryotes, the mRNA is processed by splicing out
introns and protecting the ends. These events do not occur
in prokaryotes.
Transcription Control
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The key event is binding of RNA polymerase to the
promoter. It is affected by several factors.
– promoters vary slightly in sequence, and these
variations affect the strength of binding.
– RNA polymerase has a subunit called sigma. There
are several different sigma factors in the cell, each of
which is specific to a different class of promoter. This
provides large-scale gene regulation.
– genes are controlled individually by the binding of
regulatory proteins, called transcription factors, to
regions near the promoter.
• Some transcription factors block transcription and others
encourage it
• Some transcription factors regulate whole groups of
genes scattered throughout the chromosome: a regulon
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Lac operon model (Jacob and Monod).
– Gene used to convert the sugar lactose into glucose,
which is used as food.
– The repressor protein (transcription factor) binds to
DNA (the operator) near the promoter and physically
blocks transcription.
– when lactose is present, the repressor binds to it,
changes its conformation, and falls off the DNA. This
allows RNA polymerase to bind and transcription to
proceed.
Transcription Termination
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Two basic types: rho-dependent and rho-independent.
– rho is a protein
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Rho-dependent termination involves rho protein binding
to the RNA and moving along it until it catches up with
the RNA polymerase and knocks it off the DNA
Rho-independent termination involves the newly
synthesized RNA folding up into a hairpin loop, due to
complementary bases. This sudden folding knocks the
RNA polymerase off the DNA.
– formation of stem-loops in RNA also affects transcription
initiation in some genes
mRNA, Translation, Operons
•Translation is the process of converting the
information on messenger RN.A into protein.
•Note that not all of the mRNA is translated:
there is an untranslated region of variable
length at both ends, called 5’-UTR and 3’UTR
•In bacteria, some adjacent genes with
related functions are transcribed onto the
same mRNA. This is called an operon.
•the proteins are translated individually
•this allows a fixed ratio of the proteins to be
produced.
Genetic Code
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There are only 4 bases in DNA and RNA, but there
are 20 different amino acids that go into proteins.
How can DNA code for the amino acid sequence of
a protein?
Each amino acid is coded for by a group of 3
bases, a codon. 3 bases of DNA or RNA = 1
codon.
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3 of the 64 codons are used as STOP signals; they
are found at the end of every gene and mark the
end of the protein.
In bacteria, translation usually begins at an ATG
codon, but GTG and TTG are also common start
codons.
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Since there are 4 bases and 3 positions in each
codon, there are 4 x 4 x 4 = 64 possible codons.
This is far more than is necessary, so most amino
acids use more than 1 codon.
and, a few others are occasionally used
In all cases, the first amino acid is N-formyl
methionine, a derivative of the normal methionine
used within the protein.
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note that “start” codons are also used within the
protein
this makes is difficult to be sure where the protein
actually starts
Transfer RNA
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Transfer RNA molecules act as adapters between the
codons on messenger RNA and the amino acids.
Transfer RNA is the physical manifestation of the
genetic code.
Each transfer RNA molecule is twisted into a knot
that has 2 ends.
At one end is the “anticodon”, 3 RNA bases that
matches the 3 bases of the codon. This is the end
that attaches to messenger RNA.
At the other end is an attachment site for the proper
amino acid.
A special group of enzymes (aminoacyl tRNA
synthetases, which are highly conserved in
evolution) pairs up the proper transfer RNA
molecules with their corresponding amino acids.
Transfer RNA brings the amino acids to the
ribosomes, which are RNA/protein hybrids that
move along the messenger RNA, translating the
codons into the amino acid sequence of the
polypeptide.
Translation
• Three main players here:
messenger RNA, the ribosome,
and the transfer RNAs with
attached amino acids.
• First step: initiation. The
messenger RNA binds to a
ribosome, and the transfer RNA
corresponding to the START
codon binds to this complex.
Ribosomes are composed of 2
subunits (large and small),
which come together when the
messenger RNA attaches during
the initiation process.
– there are also several protein
“initiation factors” that assist
in this process
More Translation
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Step 2 is elongation: the ribosome
moves down the messenger RNA,
adding new amino acids to the growing
polypeptide chain.
The ribosome has 2 sites for binding
transfer RNA. The first RNA with its
attached amino acid binds to the first
site, and then the transfer RNA
corresponding to the second codon bind
to the second site.
The ribosome then removes the amino
acid from the first transfer RNA and
attaches it to the second amino acid.
At this point, the first transfer RNA is
empty: no attached amino acid, and the
second transfer RNA has a chain of 2
amino acids attached to it.
Translation, part 3
• The ribosome then
slides down the
messenger RNA 1
codon (3 bases).
• The first transfer RNA
is pushed off, and the
second transfer RNA,
with 2 attached amino
acids, moves to the
first position on the
ribosome.
Translation, part 4
• The elongation cycle
repeats as the
ribosome moves down
the messenger RNA,
translating it one
codon and one amino
acid at a time.
• Repeat until a STOP
codon is reached.
Translation, end
• The final step in translation is
termination. When the
ribosome reaches a STOP
codon, there is no
corresponding transfer RNA.
• Instead, a small protein called a
“release factor” attaches to the
stop codon.
• The release factor causes the
whole complex to fall apart:
messenger RNA, the two
ribosome subunits, the new
polypeptide.
• The messenger RNA can be
translated many times, to
produce many protein copies.
Post-translation
• The new polypeptide is now floating loose in the
cytoplasm. It might also be inserted into a membrane, if
the ribosome it was translated on was attached to the
membrane by a special RNA/protein hybrid molecule.
• Polypeptides fold spontaneously into their active
configuration, and they spontaneously join with other
polypeptides to form the final proteins.
• Sometimes other molecules are also attached to the
polypeptides: sugars, lipids, phosphates, etc. All of these
have special purposes for protein function.