I. Major Players in Translation
A. messenger RNA (eukaryotes)
1. Cap
at 5' end
2. 5'
untranslated region
3. AUG
start codon
4. open
reading frame (coding region)
5. 3'
untranslated region
6. poly
A tail
B. transfer RNA
1. stem-loop
structures
2. anticodon-loop
a. codon-anticodon base pairing
b. wobble position
c. conncection between wobble and the degeneracy of the
genetic code
3. amino
acid attachment to 3'end
C. amino-acyl-tRNA synthetase
1. catalyzes
attachment of amino acid to either the 2' or 3' carbon of the ribose of
the adenine
at the 3' end of tRNA molecule.
2. two
step reaction
a. Pyrophosphate removed from ATP; AMP attached to amino
acid through COOH
group.
b. AMP removed and amino acid attached to the tRNA.
3. Specificity
of the enzyme assures the correct attachment.
D. Ribosomes
1. Two
subunits, large and small
2. Consist
of ribosomal RNAs and ribosomal proteins.
3. Contain
the enzymatic activity needed during protein synthesis.
4. Function
in the cytoplasm.
II. The Translation Mechanism
A. Initiation
1. Sets
the reading frame.
2. Small
subunit, initiator tRNA, eIF2- GTP, and other initiation factors interact.
3. Other
initiation factors bind to the Cap on mRNA and to the 5' untranslated region
4. complex
(step 2) interacts with factors at the Cap.
5. Factors
at the cap and at the 5' untranslated region melt any stem-loop in the
5'untranslated region. Requires ATP.
6. Small
subunit complex scans along until it finds the first AUG. This is called
ribosome scanning.
7. Initiator
tRNA anticodon binds to AUG codon.
8. Large
ribosomal subunit joins creating the functional ribosome with a P, A and
E site.
9. eIF2-GDP
is released and binds to eIF2B. GDP is displaced and GTP binds. This
can now interact with initiator tRNA and start another cycle of initiation.
10. If
eIF2-GDP is phosphorylated, it cannot be displaced and this inhibits
translation initiation and is an example of control at the level of translation.
11. Efficiency
of translation is increased by Kozak's sequences surrounding the AUG
start codon, facilitating the recognition of AUG by the initiation complex.
12. Internal
Ribosome Entry can occur on some eukaryotic mRNAs that are under
tight control of translation (for example growth factors, etc.).
a. IRES sequences just upstream of an AUG are recognized
by the initiation
complex which then moves to the AUG and initiates there.
B. Elongation
1. eEF1
bound to GTP binds to tRNA.
2. This
complex moves to the A site of the ribosome.
3. If
the anticodon on the tRNA is correct for the codon, it is held there long
enough for
4. GTP
to be hydrolyzed to GDP causing the eEF1 to disconnect from the tRNA and
leave
5. The
energy released in step 4 stably attaches the tRNA to the A site.
6. Peptidyl
transferase activity (probably rRNA in the ribosome) catalyzes the breaking
of
the bond holding methionine to the initiator tRNA and using the energy
released to
attach the methionine to the amino acid attached to the tRNA at the A site
by a
peptide bond.
7. With
the help of eEF2 and GTP, the ribosome moves one codon along the mRNA so
that the tRNA is now at the P site and the A site awaits another tRNA complex.
This is called translocation.
8. The
process repeats itself over and over until a stop codon is at the A site.
9. The
eEF1 bound to GDP (see step 1) is regenerated to the GTP bound form by
eEF1b binding to the GDP bound form, causing its displacement and allowing
the GTP
to bind. It can now interact with another tRNA.
10. This mechanism provides
a kinetic proofreading that insures that only the correct tRNA
will remain at the A site long enough to allow the above events to happen
before a
peptide bond is made.
C. Termination
1. When a stop codon is
at the A site of the ribosome, release factors (RF1 or RF2 bind
there. They interact directly with the stop codon.
2. Peptidyl
transferase breaks the attachment between the polypeptide and the tRNA
at the
P site.
3. The
polypeptide is released with an intact COOH terminus; the tRNA at the P
site exits
and with the help of RF3-GTP, hydrolyzing GTP to GDP, the entire ribosomal
complex
comes apart.
D. Additional Considerations
(E site, etc,)
1. The
ribosome also has an E site where the exiting tRNA moves during translocation.
2. The
tRNA orientation with the ribosome during translation begins with P/P.
3. Then
a tRNA enters the A site first with its anticodon (A/T orientation)
4. Then
the amino acid side binds (A/A orientation)
5. After
peptide bond formation, both tRNAs lean over such that the P site tRNA
has its
3' end at the E site (P/E orientation) and the A site tRNA has its amino
acid end at the
Psite (A/Porientation).
6. Following translocation,
the previous P site tRNA is now attached to the E site only by its
3' end and will soon leave completely. (E orientation). And at the P site
now is the tRNA
that had been at the A site, completely attached (the P/P orientation).
E. Additional events of note
1. Efficiency
of translation is increased because the poly A tail of the mRNA binds
poly-Abinding
protein 1 (PAB1). This interacts directly with the eukaryotic initiation
factor
at theCap,
facilitating the entry of the small ribosome initiation complex onto the
mRNA to
start a new round of initiation.
2. Another
eIF binds to the large ribosomal subunit, preventing it from reuniting
with the
small subunit until initiation has been completed.
F. Prokaryotic Differences
1. Initiation
and setting of the reading frame can occur in more than one place (polycistronic)
a. uses Shine-Delgarno interactions between mRNA codons
before the AUG interacting
with ribosomal RNA sequences.
G. Control at the level of translation
a. The ferritin mRNA translation is controlled at the
level of initiation.
-The stem-loop in the 5'UTR contains an Iron Response Element that can
be bound by
the IRE binding protein if concentrations of iron are low in the cell.
-This prevents the small subunit inititation complex from scanning along
to find AUG after
the Cap.
-Translation cannot be initiated.
-If, however, iron concentrations are high, the ferritin should be made
to store the excess
iron and the IRE binding protein will not be able to bind and the stem-loop
can be
melted. Translation can be initiated.
b. Another
translational control involves eIF2 which can be phosphorylated to prevent
it from becoming active (bound to GTP).
-The eIF2B protein cannot displace the bound GDP if eIF2 is phosphorylated.
-since eIF2-GTP is needed to make the ternary complex that is used during
translation initiation, this prevents initiation.
III. Protein Function
A. Chaperones and Chaperonins
1. Chaperone
proteins bind to exposed hydrophobic side chains on proteins to prevent
their aggregation until the protein is fully translated.
a. They then use the energy of ATP to release from the
protein which then can
fold correctly.
b. They also function to help denatured proteins refold
correctly.
2. Chaperonins
are needed to help some proteins fold correctly.
a. They sequester the partially folded protein inside
a barrel-like structure until
the protein is correctly folded.
b. They then release it, using the energy of ATP.
B. Protein degredation
1. Some
short-lived proteins are marked for degredation by sequences that are recognized
by ubiquitinylating enzymes.
a. These enzymes attach a polymer of repeating ubiquitins
to the amino group attached
to the side chain of lysine residues in the protein.
b. Other factors then bind these ubiquitins and bring
the protein to a proteosome.
c. Within the core of the proteosome, the protein is
degraded by proteases.
2. Short-lived
proteins versus longer-lived proteins have different amino acids at their
amino terminal.
3. Also,
some short-lived proteins often contain PEST sequences (proline, glutamic
acid,
serine, threonine).
C. Importance of 3-D structure to protein
function
1. The
3D structure of proteins allows the proper chemistry at the bindins site
or active
site of proteins.
2. Our example was the cyclic-AMP
dependent protein kinase.
a. When bound by cAMP, the two regulatory subunits disengage
from the catalytic
subunits.
b. The catalytic subunits catalyze the attachment of
phosphate to proteins on a serine
or threonine.
c. Only proteins with a specific recognition sequence
for the catalytic subunit will be
phosphorylated.
d. The active site of the enzyme binds both ATP and the
polypeptide.
e. This causes an induced fit, bringing the polypeptide
closer to the ATP. (Closed)
f. Also causes a glycine lid to move over the ATP binding
pocket to keep it there.
(Closed conformation)
g. The chemistry at the active site allows for the creation
of a pentavalent gamma
phosphate transition state. This is achieved because the phosphate group
electrons are dispersed by attraction to either lysine side chains or Mg+2
cofactors at the active site. One Mg+2 is held by interaction with an
aspartic acid at the active site. Also, a very important aspartic acid
at the active site
pulls the H from the OH group of the serine or threonine, causing the O
to interact
with the gamma phosphate, leading to the transition state. See page 72.
The
beta-gamma phosphate bond breaks ate the gamma phosphate remains attached
to the serine or threonine side chain.
h. Upon formation of the products, the conformation of
the enzyme returns to its
original shape (Open), allowing ADP to be released and the phosphorylated
polypeptid to leave.
D. Allosteric regulation
1. Allosteric
enzymes have both regulatory and catalytic subunits.They exist in either
active (R) or inactive (T) configurations that are in equilibrium unless
bound by regulators.
2. When
regulators bind to the regulatory subunits of one form, that form is stabilized.
3. Example:
aspartate transcarbamoylase.
a. Works early in the pathway leading to pyrimidine synthesis.
b. Can be bound by CTP, a negative regulator.
c. When the inactive form is bound, it is stabilized
and cannot revert to an active form.
d. This decreases the number of active forms thus decreasing
the rate of the reaction
catalyzed by the enzyme.
e. CTP is an end-product of pyrimidine biosynthesis and
therefore can feedback and
regulate the pathway that produced it. Called feedback inhibition.
E. Other controls
a. Proteins
that are multi-subunits and have symmetry can show cooperativity of ligand
binding. The binding of one ligand induces a conformational change in the
other
symmetrical sites, allowing subsequent ligans to bind to them more easily.
IV. Signal Transduction
A. Three overall types
1. Endocrine:
hormone made far away and brought through blood to target cell.
2. Paracrine:
Cells are adjacent. Signal secreted from cell or part of its membrane.
Then
interacts with target cell next to it.
3. Autocrine:
Cell that makes the signal has receptors itself for that signal.
B. G stimulatory system
1. Receptor
changes shape upon activation by signal.
2. Binds
to G stimulatory.
3. Causes
alpha subunit (bound to GDP) to disengage form beta-gamma subunit.
4. Causes
GDP to leave and it is replaced by GTP.
5. Allows
alpha subunit to bind and activate adenylate cyclase.
6. Adenylate
cyclase catalyzes formation of cyclic AMP from ATP.
7. When
hormone no longer bound to receptor, alpha subunit hydrolyzes GTP to GDP
causing reversal of the above events and a return to the beginning.
8. Cyclic
ATP binds cyclic AMP-dependent protein kinase.
9. Causes
regulatory subunits to displace from catalytic subunits, activating them.
10. Phosphorylation
of substrates alters their activities, transducing signals.
11. G
inhibitory is similar to G stimulatory with the difference being that the
alpha
subunit inhibits adenylate cyclase activity thus lowering cyclic AMP levels.
C. Gp system (phosphatadyl inositol
system)
1. Receptor
binds signal and activates phospholipase C.
2. Phospholipase
C cleaves PIP2 in the membrane.
3. Produces
Diacylglycerol (DAG) which remains in the membrane and activates
protein kinase C.
4. Also
produces InP3 which is released into the cytoplasm and binds to calcium
channel
in the ER membrane, causing the release of calcium into the cytoplasm from
the lumen.
5. InP3
will recycle back to PIP2. Some cells use an alternate pathway that phosphorylates
it to a tetraphosphate intermediate that can bind calcium channels in the
cell membrane
and allow extracellular calcium to enter the cell.
6.
Calcium binds to calmodulin. This complex binds to other proteins and alters
their
activity, transducing the signal.
D. Receptor tyrosine kinase system
1. Receptor
tyrosine kinases are transmembrane proteins that are single proteins until
activated by a signalling ligand.
2. When
bound, they dimerize with identical receptor tyrosine kinase.
3. They
autophosphorylate their cytoplasmic catalytic domains at tyrosines (each
on the
other).
4. Proteins
with SH2 domains bind to the phosphotyrosines.
5. Proteins
with SH3 domains bind these or, alternatively, the SH2 domain protein may
itself have an SH3 domain.
6. SH3
domains bind other proteins activating them and transducing the signal.
7. Ras
is one protein that is so activated.
8. Ras
bound to GDP is inactive. When a GEF protein binds it the GDP is displaced.
9. GTP
binds and ras can then bind other proteins and activate them.
10. Ras
binds to GAP which accelerates ras's GTPase activity and GTP is hydrolyzed.
E. Epidermal Growth Factor system
1. EGF
binds EGF receptor (a receptor tyrosine kinase).
2. The
receptor dimerizes and autophosphorylates.
3. GRB2
binds the receptor through its SH2 domain (has both SH2 and SH3 domains).
4. Sos
binds to the SH3 domain and is activated.
5. Sos
(a GEF) binds ras causing it to displace GDP, thus actiavating ras (see
above).
6. Ras
activates raf by binding to it.
7. Raf
(a serine-threonine kinase) phosphorylates MEK, activating it.
8. MEK
(a serine-threonine and a tyrosine kinase) phosphorylates MAP Kinase.
9. MAP
Kinase moves into the nucleus and activates the transcription of growth-regulatory
genes.
V. Apoptosis
A. 2 types of cell-death.
1. Necrosis:
due to trauma.
a. cells lyse and die
2. Apoptosis
a. programmed cell-death
b. cells commit suicide
B. Trophic Factors
1. Survival
signals that must be present to prevent apoptosis
2. Examples:
Nerve growth factor in the developing nervous system
a. called a neutrophin
b. activates a receptor tyrosine kinase called Trk
C. Apoptosis Mechanism
1. Identified
proapoptotic factors (CED 3, 4) and anti-apoptotic factors (CED 9) in
elegans
2. Human
equivalent of CED 9 is bcl-2
a. protein locted in the outer mitochondrial membrane,
nuclear membrane, and ER
membrane.
D. Effectors of the apoptosis
pathway
1. Caspases
a. cysteine proteases that cleave to the COOH side of
aspartic acid residues in target
proteins.
E. Pro-Apoptotic Factors
1. Bax
a. in the bcl2 family but promotes apoptosis rather than
inhibiting it.
F. Cytochrome c involvement
1. Usually located
between the inner and outer mitochondrial membranes.
2 Released
to cytoplasm during apoptosis.
3. Binds Apaf-1
(mammalian equivalent of CED-4).
4. Initiates
a caspase cascade.
G. Bax/Bax homodimers in the outer
mitochondrial membrane form an ion channel allowing
influx of ions. Bax/Bcl2 or Bcl2/Bcl2 heterodimers do not.
1. Could
cause the release of cytochrome 3 into the cytoplasm.
H. Trophic Factors induce inactivation
of the pro-apoptotic regulator, Bad.
1. Induce
its phosphorylation (probably by P1-3 kinase activating Akt kinase).
2. Phosphorylated
Bad binds to 14-3-3 (a phosphoserine binding protein) in the cytoplasm.
3. Therefore
it cannot bind to the Bcl2/Bclxl dimer in the outer mitochondrial membrane.
4. If
Bad does not bind, the Bcl2/Bclxl dimer can inhibit the Bax/Bax formation
of the ion
channel.
5. This
prevents release of cytochrome c and the apoptotic events outlined below.
I. The apoptotic pathway
1. Bad
is bound to bcl2/bclxl dimer.
2. The
dimer cannot inhibit the Bax/Bax homodimer
3. It
forms an ion channel and ions enter
4. cytochrome
c is released into the cytoplasm
5. cytochrome
c binds to Apaf1
6. Apaf1
activates procaspase 9
7. Procaspase
cleaves itself forming caspase 9
8. Caspase
9 cleaves procaspase 3 forming caspase 3
9. Caspase
3 cleaves multiple substrates ultimately causing cell death.
VI. Cancer
A. Oncogenes
1. Formed
from proto-oncogenes
a. Normal cellular proteins that have become deregulated
in some way.
b. Are dominant, gain-of-function mutations or changes.
2. Originally
studied in retroviruses that were oncogenic.
a. viral src protein loses a tyrosine that usually becomes
phosphorylated to turn the kinase
activity of the src protein off.
b. SH2 domain of c-src binds this phosphorylated tyrosine
and causes a shape change
in thekinase site.
c. viral src was transduced from a host cell during retroviral
replication.
d. Retroviruses usually have gag (capsid protein), pol
(reverse transcriptase), and env
(spike proteins in the lipid envelope) genes but oncogenic forms have an
onc
gene also. For example, v-src.
e. Retroviruses are RNA viruses that go through a ds
DNA intermediate that integrates
into the host cell's chromosomes.
3. Human
counterparts and other human proto-oncogenes identified by transfection
assays
using normal mouse cells.
a. DNA from human cancer is transfected into the mouse
cells and transformed foci are
allowed to develop if possible.
b. Foci DNA is extracted and used for a second round
of transfection.
c. Foci DNA from second round is shotgun cloned into
a phage library that is used to
infect bacteria.
d. Plaques are relica plated and probed with human Alu
sequences
e. These spacer sequences flank all human genes and itentify
the presence of human DNA
in the phage.
f. These phage are the source of DNA to study the human
oncogene identified.
g. Mutant ras identified in this way. Cannot hydrolyze
its GTP and is therefore always on.
4. Seven
categories of growth-control proteins can be involved in malignant transformation
if
altered.
a. Category I is growth-factors. Rare. sis oncogene is
a truncated version of PDGF that
interacts with incorrect receptors.
b. Category Ia are viral proteins that can activate receptors
they should not interact with.
c. Category II are intracellular receptors that can activate
transcription even in the absence
of the soluble signalling hormone binding to them and cell-membrane receptors
that are
constitutively active.
-example: Her-2 receotor has a valine to glutamine point mutation, creting
the
neu oncogene that activates its receptor tyrosine kinase activity by causing
dimerization
and autophosphorylation on tyrosine.
-example: EGF receptor can be truncated, forming the erbB oncogene. Truncation
causes
the removal of the extracellular domain, constitutively activating the
kinase
domain causing dimerization and autophosphorylation on tyrosines.
d. Category III is a large category of all intracellular
signalling molecules. (Ras and src are
in this category). Can also include some tumor-suppressor proteins as well
as proto-
oncogenes.
- anothere example is the tyrosine kinase c-abl. Becomes oncogenic when
it fuses with the
bcr gene due to a reciprocal translocation between chromosomes 9 and 22.
-creates the Philadelphia chromosome, containing the abl gene from 9 attached
to the bcr
gene on 22. abl kinase activity is activated.
-causes chronic myelogenous leukemia in children.
e. Category IV are transcription factors
-examples are fos and jun which are activators of transcription initiation.
-another example is myc which is a transcription factor that is often deregulated
by
gene amplification.
--detected by the presence of HSR or double-minutes on chromosome
spreads indicating multiple copies of the gene.
--can also be activated by translocation between chromosomes 8 (myc) and
14
(antibody genes). The powerful antibody gene enhancers abnormally activate
the translocated myc.
f. Category V are anti-apoptosis factors
-examples are survival factor receptors that get constitutively activated.
Example: trk
--trk is activated by a translocation that replaces its amino-terminal
region with
sequences from the non-muscle tropomyosin protein.Causes it to dimerize
and
autophosphorylate on tyrosines, transducing the survival signals that activate
anti-apoptotic factors.
-others are proteins like bcl2 which inhibit bax homodimers from opening
ion channels.
-any anti-apoptotic protein that is abnormally active is in this category.
g. Category VI are Cell-cycle control proteins that promote
movement through the cell-
cycle. Examples: cyclins, cdks, mdm-2. Tumor-suppressors like p16, p27,
and
p21 also work at this level.
h. Category VII are tumor-suppressor protein involved
in DNA repair mechanisms.
i. Although the categories contain tumor-suppressors
also, these are not oncogenes (see
below).
j. Telomerase is another potential oncogene in that its
continual expression is correlated
with maintaining the normal lengths of telomeres in cancer cells.
-Could extend and immortalize the cell
-Normal cells have not telomerase and undergo senescence (stop dividing).
-mouse models do not support this idea however but it could be different
in humans.
k. Oncogenes can also become activated by insertional
mutagenesis whereby a retrovirus
integrates near one of the growth promoting genes and activates it due
to the powerful
enhancers it carries. (Example: int gene should only be active in development
but is
activated by retroviral integration).
B. Tumor-Suppressor Proteins
1. Are
recessive.
2. Must
lose both copies to have an effect.
3. Involved
in inhibiting growth signals, inhibiting progression of the cell-cycle,
in DNA repair,
in inducing apoptosis, and in causing cell-cycle arrest.
4. Some
are in Category III.
a. NF1 is a mutated GAP that cannot help ras hydrolyze
GTP. The GAP therefore is a
tumor-suppressor protein.
b. PTEN is a protein phosphatase that removes phosphates
from serine, threonine, and
tyrosines, thereby shutting off growth signals.
5. Many
are Category VI proteins.
a. p16, p27, and p21 all bind to cyclin/cdk complexes
and thereby prevent them from
phosphorylating their target proteins.
b. One target is the RB (retinoblastoma) protein which
when not-phosphorylated binds to
a transcription factor family called E2F and prevents the activation of
genes involved
in further cell-cycle progression, usally from G1 into S.
-Cdk4/cyclinD and Cdk6/cyclinD phosphorylate Rb in mid-G1; cdk2/cyclinE
in
late G1. These cyclins are activated in response to mitogenic stimuli.
c. RB itself is a tumor-suppressor protein since it prevents
cell-cycle progression when
bound to E2F.
d. A receptor protein, TGF-beta is a tumor-suppressor
that can regulate cell-cycle
progression by phosphorylating Smads at serines and threonines.
-Smad2 or Smad3 will be activated by this and can bind Smad4.
-The complex moves to the nucleus and activates genes that inhibit proteases
(PAI-1),
preventing metastasis or genes that inhibit cell-cycle progression (e.g.
p15).
-The Smads are also considered to be tumor-suppressor proteins.
e. p53 is a tumor-suppressor protein involved in multiple
ways.
-when induced by events that cause damage to DNA, e.g. irradiation, it
can cause the
cell-cycle to arrest by inducing the transcription of p21, a cyclin-cdk
inhibitor.
f. It also can induce apoptosis in cells alternatively,
especially if the DNA damage is
extensive.
g. It therefore helps maintain the integrity of the genome
of the cell, preventing the
accumulation of genetic damage that would occur if cells were allowed to
divide in the
presence of such damage.
h. Some p53 mutations are dominant negative, causing
a loss of p53 function even in
the presence of one functional allele.
-due to the fact that p53 functions as a tetramer and some mutant subunits
can cause
the tetramer to misfunction.
i. mdm-2 is a proto-oncogene product that binds to and
inactivates p53.
-p53 becomes phosphorylated by several protein kinases in response to DNA
damage.
-mdm-2 can no longer bind. This releases active p53.
-p53 activates transcription of mdm-2 gene, thus autoregulating its own
activity.
6. Some
are Category VII proteins that are directly involved in DNA repair mechanisms.
a. nucleotide-excision repair proteins and mismatch-repair
proteins have
been identified as lost in many cancers.
7. Some
are Category V proteins that are in the pathway that leads to apoptosis.
This can
also include p53.
8. Several
DNA Tumor Viruses bind to these tumor-suppressor proteins to inactivate
them
when the virus transforms cells.
a. Example: SV40 which binds both to
p53 and to Rb through its large T antigen.
b. Example: Adenovirus which binds
p53 through its E1b antigen and Rb through its
E1a antigen.
c. Example: Human Papilloma Virus (HPV)
which binds to Rb through its E7 antigen
and binds to and causes the degredation of p53 through its E6 antigen.
C. Other Cancer-related events
in the cell
1. Angiogenesis
contributes to tumor-growth
a. This formation of new blood vessesls to feed the tumor
is promoted by factors secreted
from the tumor. Example: VEGF
b. Anti-angiogenic factors show promise of inhibiting
this blood-vessel formation, thus
starving the tumor. example: endostatin
c. Primary tumors secrete inhibitors of angiogenesis
around secondary tumor sites!
2. Cancer
is a multi-step process.
a. Roughly 5-6 genetic changes in growth-control pathways
must occur to form a highly
aggressive cancer in humans.
b. Both oncogene activation and tumor-suppressor loss
must occur.
c. The latest stages involve metastasis-related mechanisms
that are poorly understood but
allow transformed cells to break free from their tissues and move to other
sites.