I. Transcription
A. Chemistry of the reaction to
make an RNA polymer
1. Use
NTPs as energy source. Remove Pyrophosphate.
2. Attach
remaining monophosphate in a 5'-3' linkage, similar to DNA linkages.
B. RNA Polymerases
1. Do
not need a preexisting 3'OH. Can attach two nucleotides together to
start
an RNA
polymer.
2. Types
a. RNA pol I: transcribes the large rRNAs in
eukaryotes
b. RNA pol II: transcribes the primary RNA transcript
that
will become mRNA in eukaryotes
c. RNA pol III: transcribes small RNAs in eukaryotes
(tRNAs, etc.)
3. Contain
three activities
a. unwinding activity (helicase): allows the ds DNA
to
become single-stranded.
b. polymerizing activity: forms the
RNA transcript.
c. rewinding activity: displaces
the
RNA transcript (about 10-15 nucleotides long) from the template strand
and allows the ds DNA to reform.
C. Regulatory Sequences
1.
Promoter
a. Loads RNA polymerase II onto the DNA where it can
then find the startpoint of
transcription.
b. Located approximately 30-50 nucleotides upstream
of
the +1 startpoint.
c. Most common type is the TATA box.
-Consists of a consensus sequence TATAA/TA that is the site of assembly
of the
general transcription factors.
d. Alternative promoter types exist but are not
included
on the exam.
2.
Upstream
Promoter Elements
a. Located approximately 200-500 basepairs upstream
from
+1 startpoint.
b. Many different types have been identified.
c. Bind the specific transcription factors.
d. Believed to modify the ability of the general
transcription
factors to load on RNA
pol II.
e. Bind both activators and/or repressors.
-activators can be histone acetylases, acetylating histones at their
amino
termini, loosening
the chromatin structure or chromatin remodeling factors that phase
nucleosomes away from transcription factor binding sites. Histone
chaperones can
also modify chromatin by
removing histone cores or replacing histone cores in chromatin
structures.
-activators can directly bind other cofactors and make it easier for
the
general
transcription factors to bind at the TATA box.
Often bind to mediator complex of proteins.
-repressors can be histone deacetylases. Deaceylated histones are more
compactly packed.
-repressors can compete with activators for binding sites or suppress
their
activation
domains by binding to them.
-repressors can directly bind to the general transcription factors and
inhibit further
assembly.
3.
Enhancers
a. Sequences that can modify transcription
rates
at large distances from the +1
startpoint (1-5kb) and on either side of the gene and in either
orientation.
Sometimes found in introns.
b. Bind additional specific transcription factors
that
can either upregulate or downregulate by all the methods described
above.
c. Believed to interact with the general
transcription
factors by looping the DNA
to bring the enhancer(silencer) binding proteins to the vicinity of the
promoter.
d. Interact with chromatin remodeling factors that
cause nucleosomes to "slide" so that histone modifying enzymes
can gain access to them.
e. Often interact with mediator (giant complex of proteins)
which itself binds to activation domains of specific transcription
factors.
4. How
RNA polymerase gets loaded on:
a. In-vitro studies indicate the following mechanism.
b. TBP (TATA binding protein) binds to the TATA.
(Part of the larger TFIID which also contains many tafs)
c. The remainder of the general transcriptions
assemble.
d. This includes TFIIF bound to RNA polymerase II.
e. Once all are assembled, TFIIH acts.
-Its protein kinase activity attaches phosphates to RNA polymerase II
at
its CTD
-Its helicase activity melts the hydrogen bonds of the DNA to expose
the
template.
f. RNA polymerase II will begin transcription and
clear
away from the promoter region.
g.
in-vivo,
A large complex called mediator interacts with the TATA box region
factors
and forms a
bridge between these factors and specific transcription factors, often
bound to enhancer elements.
D. Many eukaryotic genes are
regulated
at the level of transcription initiation.
E. Antitermination
1. RNA polymerase II often pauses during elongation
of
transcription and will only
complete transcription when signalled to do so.
2. One example is the tat protein of HIV.
- Tat binds to a sequence near the 5' end of the transcript called TAR,
part of a stem-
loop structure.
-Cellular factors bind to another stem-loop structure.
-Together they coordinate interaction with other cellular proteins that
includes
cdk9, a protein kinase that phosphorylates the CTD of the stalled RNA
pol
II and
causes it to continue transcription.
3. Another example is the control of the hsp70
transcript
which responds to stress in cells.
-The non-stressed cell begin transcription but the RNA polymerase II
pauses.
-In response to stress, a protein called HSTF changes shape from an
inactive
to an
active form.
-It now can bind near promoter-proximal elements called GAGA.
-In some way this causes the stalled RNA polymerase II to resume
transcription
as
well as stimulating more rounds of transcription by other RNA
polymerase
IIs.
4. The CTD
phosphorylation at ser 5 in the repeats allows the capping enzymes to
bind. Additional phosphorylations at ser 2 allow the cleavage and
polyadenylation factors to bind and many splicing factors as well.
F. Processing the primary
RNA transcript
1.
Capping
the 5' end.
a. The CAP is a G nucleotide methylated at the N7
position
of the base and
attached to the first nucleotide of the RNA transcript by a 5'-5'
triphosphate
bridge. In vertebrates, the 2'O of the ribose is methylated in the
first
and second
nucleotides also.
b. This CAP is used during the initiation of
translation
to help set the reading frame, participates in nuclear
export, and protects the growing transcript in the nucleus.
c. First, capping enzymes remove the gamma phosphate
from the 5'nucleotide of the transcript.
Then, GTP has Pyrophosphate removed and the GMP is attached to the 5'
nucleotide
by the 5'
-5' triphosphate bridge. Then the 7N of the guanine is methylated by
methylase
enzymes and the 2'OH is
of the first nucleotide of the transcript is also methylated. In
vertebrates,
the second nucleotide is also
methylated in this manner. S-adenosyl methionine (SAM) is the source of
the methyl groups.
2.
3'-end processing (cleavage and polyadenylation)
.
a. In the primary transcript, a poly-adenylation signal is
made that reads AAUAA.
b. The actual cleavage and poly-A addition site is about
20-30
nucleotides downstream.
c. RNA pol II continues transcription past these
sites
and makes a G/U rich sequence in
the transcript. It continues transcribing.
d. CPSF binds to the AAUAA signal.
e. This facilitates the binding of the cleavage factors and
CstF, which binds
to the G/U-rich sequence.
f. This allows PAP (poly-A polymerase) to bind.
g. The cleavage factors cleave the transcript at the poly-A
site and PAP attaches the
poly-A tail.
h. At first the poly-A addition is slow.
i. PABII (a poly-A binding protein binds to the poly-A tail and
rapid
polyadenylation proceeds. About 200 A
residues get attached. The rest of the transcript beyond the cleavage
site
is degraded.
3. Removal of
introns
a. Eukaryotic genes contain intervening sequences called
introns.
b. RNA polymerase II transcribes these introns.
c. They must be removed from the transcript and the
exons
(what is left) connected into a
continuous mRNA sequence.
d. This is done by a mechanism known as splicing.
e. Evidence for splicing comes from a technique known as
R-looping
-mRNA is hybridized to single-stranded DNA strands (hydrogen bonds have
been melted) from the same cell.
-Complementary sequences will hybridize.
-Intron sequences will have no complementary sequence in the mRNA.
-They "loop out" and can be seen in electron micrographs.
f. The splicing signal in the primary transcript has
three major features.
-There is a 100% conserved GU at the 5'end of the intron.
-There is a 100% conserved AG at the 3' end of the intron.
-There is an A nucleotide about 20-30- nucleotides from the 3'end of
the
intron that
is called the branch point and is 100% conserved.
A pyrimidine-rich region lies after the A and before the 3' end.
g. The intron is removed by two transesterification
reactions.
-First, the 2'OH on the ribose of the branch point A attacks the 3' end
of exon 1.
Tthis breaks the phosphodiester bond between the last nucleotide of
exon
1 and the
first nucleotide of the intron.
-A bond forms between the 5'P of the first intron nucleotide and the
2'OH
of the A.
-Then, the 3' end of exon 1 (the 3'OH) attacks the 5' end of exon 2.
-This breaks the bond between the 3' end of the intron and the 5'end of
exon 2.
-The 3' end of exon 1 links to the 5'end of exon 2 by a phosphodiester
linkage.
-The intron (in lariat form) is removed and degrades.
h. These reactions are catalyzed and controlled by
nuclear
particles called snurps.
-consist of small nuclear RNAs complexed to proteins.
-several kinds are involved, called U1, U2, etc.
-U1 has snRNA that is complementary to the GU and flanking sequences at
the exon/
intron junction. It binds first during the splicing reaction. Then BBP
binds the branch-point A region and U2AF binds the pyrimidine rich
region.
-U2 has snRNA complementary to the consensus sequence surrounding the A
branchpoint but not the A itself which bulges out. This facilitates the
first reaction.
U2 is helped onto its binding site by U2AF which binds in the
pyrimidine-rich region. U2 displaces BBP when
it binds. Thus, the branch point A site is checked out twice.
-After U2 binds, the remaining snurps bind forming the spliceosome.
-The RNA-RNA interactions between the transcript and the snurps and
between
the
snurps rearrange, using ATP hydrolysis as an energy source.
U1 is displaced by U6, thus checking the 5' splice site twice.
-Finally, the spliceosomes, still attached to the intron, leave at the
end of the splicing
reactions and the snurps disassemble.
-SR proteins bind to ESE (exonic splicing enhancers) and interact with
splicing factors
within introns on either side.
-Alternative splicing is often used in cells to make different forms of
a protein from the same transcript.
i. Sex determination in Drosophila is an example.
-Early promoter of sxl gene used in females, not males. Makes early
form
of sxl.
-Later promoter of sxl used in both males and females. Early sxl in
females
binds to a U2AF binding site near the 3' splice junction
and prevents that intron being removed; the next 3' junction is used
instead,
removing everything, including
the exon. This does not happen in males, therefore the exon remains. It
has a stop codon in-frame,
causing premature termination of translation and a non-functional sxl.
Females make the functional
sxl.
-The functional sxl in females regulates the splicing of the tra gene
transcript
in a similar fashion, causing
the removal of an exon that has a stop codon. Males do not remove this
exon, therefore they make
non-functional tra protein.j
-Female tra forms a complex with tra2 and Rbp 1. This works as an SR
protein,
binding the exon next to a 3'
splice junction, activating it. This causes inclusion of an exon and
translation
creates the female form of
double-sex protein. This represses male-specific gene transcription
leading
to the female phenotype. Males
are not able to enhance the splice junction, thus the exon is removed
as
part of a larger intron, and
translation forms the male double-sex protein which represses
female-specific
genes and the male
phenotype is formed.
G. Control of Gene Expression
at the level of 3' end processing
1.
Choice of cleavage and polyadenylation site can change subsequent
patterns
of
of splicing and/or translation, making different proteins or forms of a
protein.
2.
An example is the change from a membrane-bound form of antibody
molecule
in B cells before antigen
activation (retains a hydrophobic region that interacts with the
membrane)
and the secreted form of the
antibody, following activation (now has a hydrophilic region and no
hydrophobic
region). Due to
the choice of a downstream poly-A signal used in the first case and an
upstream signal used during
the latter case. The resulting transcripts result in different splicing
patterns and different sequences in the
COOH terminal of the proteins. The choice of site is cleavage site is
controlled by the ability of CStf to bind the GU-rich reason of the
transcript. At low
concentration, the sequence in
that region will not allow it to bind there. At high concentration, it
can. CStf concentration is lower before activation and
higher afteer activation, causing
the change in cleaveage/poly A site used.
H. RNA editing (Not covered in exam 2 this year).
1. A point mutation has been shown to be
inserted into some (few) eukaryotic transcripts by the deamination of a
C to a U, altering a
codon. Our example of this
was the two forms of apoliprotein B generated in hepatocytes versus
intestinal cells.
I. mRNA export out of the nucleus
1. A special SR protein is part
of the RNA
binding protein components that associate with the forming mRNA in the
nucleus. This binds in its phosphorylated
form.
2. Once the poly-A tail is added, a
phosphatase associates and removes the phosphate.
3. This allows an exporter protein to
bind.
4. It escorts the mRNA complex through
the nuclear pores into the cytoplasm.
5. Another kinase then reattaches
the phosphate to the SR, causing it to disengage from the exporter and
the mRNA.
6. Both the SR protein and the exporter
are then
imported back to the nucleus.
J. Nonsense-mediated mRNA decay:
1. When the mRNA arrives in the
cytoplasm, a ribosome begins to translate it until a stop codon is met.
2. If a EJC is present while the
ribosome is moving, it displaces it.
3. When the stop codon is reached, all
EJCs should have been removed unless the stop codon is present due to
mistakes in splicing or for other mistakes.
4. If an EJC is still present, it will
be detected by Upf proteins which bind to it and form a bridge to the
stop codon.
5. This causes a nuclease to degrade the
mRNA.
II. Genetic Code
A. Triplet code
B. Reading Frame
1. insertions or deletions cause frameshift
mutations.
C. Amino acid codons worked out
using the Nirenberg filter binding assay.
1. Synthetic trinucleotides added to cell extract
containing
all amino acids, tRNAs, ribosomes, and cofactors
needed for translation.
2. Divided into 20 tubes. One radioactive amino acid added to each
tube.
3. Mixes filtered, trapping ribosomes and any attached
trinucleotide
and tRNA charged with amino acid on
the filter.
4. If filter is radioactive, indicates that the radioactive amino acid
from that tube is that coded by the trinucleotide.
D. The code has start (sets the
translational reading frame) and stop (no tRNA exists) codons. It is
also degenerate (more than one codon for most of the
amino
acids). Degeneracy likely resulted from the problem of wobble,
described below.
III. 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. Inosine often found
in the anticodon.
b. wobble position allows incorrect base pairing
between
first anticodon base and third codon base
c. understand conncection between wobble and the
degeneracy
of the genetic code
3. amino
acid attachment to 3'end in conserved CCA sequence.
C. amino-acyl-tRNA synthetase
1.
catalyzes
attachment of amino acid to either the 2' (class 1) or 3' (class 2) OH
of the ribose of the
adenine at the 3' end of tRNA molecule.
Class 2 enzymes will then move the amino acid to the 3' position.
2. two
step reaction
a. Pyrophosphate removed from ATP; AMP attached to
amino
acid through COOH
group.
Is then transfered to a editing site on the enzyme. Released if
inaccurate.
b. AMP removed and amino acid attached to the tRNA.
Editing can happen at this point as well.
3.
Specificity
of the enzyme assures the correct attachment.
4. 20
groups of amino-acyl-tRNA synthetases, one group for each of the 20
amino
acids and tRNAs
D. Ribosomes .
1.
Functional ribosomes have two subunits, large and small. Know their S
values.
2. These
consist
of ribosomal RNAs and ribosomal proteins.
3. Contain
the enzymatic activity needed during protein synthesis as well as
participating in proper positioning, etc.
________________________________________________________Finishes
the information on exam 2. Begin below for exam 3 (2010)
IV. The Translation Mechanism
A. Initiation
1. Sets
the reading frame. Small subunit binds eIF6 (keeps it from binding
large
subunit).
2. Small
subunit, initiator tRNA, eIF2- GTP, interact.
3. Other
initiation factors (eIF4E) bind to the Cap on mRNA.
Poly-A binding proteins interact with eIF4G, looping the 3' end to the
5' end of the mRNA.
4. complex
(step 2) interacts with factors at the Cap.
5. eIf4
component and factors from the 5' untranslated region melt any
stem-loops
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.eIF6 leaves the small subunit.
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. eIF2
is a major target for controlling translation in the cell.
a. When phosphorylated, it permanently binds to
eIF2B.
This sequesters the eIF2B and does not allow
further eID2-GTP active forms to be made. Lowers translation overall.
11.
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. A truncated
eIF4G
binds to a stem-loop made
made by the IRES sequence and facilitates the attachment of the
initiation
complex.
B. Elongation
1.
eEF1alpha
bound to GTP binds to tRNA.
This bends the tRNA .
2. This
complex moves to the A site of the ribosome.
Only the anticodon site interacts. Codon/antiicodons incorrectly paired
will preferentially dissociate
(Proofreading
step 1)
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 eEF1alpha to disconnect from the
tRNA
and leave
5. The
energy released in step 4 stably attaches the tRNA to the A site.
This requires the ribosome to check out the codon/anticodon interaction
basepair by
basepair. Once enclosed over it entirely, it allows
the GTP hydrolysis step to occur (Proofreading step 2)
6.
Peptidyl
transferase activity (probably rRNA in the ribosome) then 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.
(If the codon/anticodon is incorrect, they will dissociate before this
is triggered (proofreading step 3)
7. With
the help of eEF2 bound to 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.
The exiting tRNA from
the P site transiently is at the E site. Then leaves.
This is called translocation.
8. The
process repeats itself over and over until a stop codon is at the A
site.
9. The
eEF1alpha 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.
11. The eEF1-GDP is bound
by eEF1 beta which forces out GDP. GTP replaces it to reactivate
eEF1-GTP
C. Termination
1. When a stop codon is
at the A site of the ribosome, RF1 (or 2, depending on the stop
codon)
binds
there, interacting directly with the stop codon.RF3-GTP binds the A
site
elsewhere.
2.
Peptidyl
transferase breaks the attachment between the polypeptide and the tRNA
at the
P site. Water enters.
3. The
polypeptide is released with an intact COOH terminus; the tRNA at the P
site exits
and RF3 hydrolyzing GTP to GDP. The entire ribosomal
complex comes apart.
D. 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 the
Cap, facilitating the entry of the small ribosome initiation complex
onto
the mRNA to
start a new round of initiation.
E. 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.
F. 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 aconitase 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. Overall
translation can be stopped by phosphorylation of eIF2-GDP, preventing
it from
being able to release from eIF2B (its GEF). Since there is less eIF2B
than eIF2 in the cell, this sequesters all the eIF2B, preventing it
from
interacting with other eIF2 molecules. Those then
cannot be reactivated to their GTP-bound state.. No new
translation
initiation complexes can form.
c. We also reviewed the
mTOR pathway and its role in overall translation control.(In 2010, this
was done during apoptosis. )
d. We also reviewed the
influence of miRNAs on translation inhibition and mRNA stability and of
siRNAs on RNA
cleavage achromatin compaction..
1.
miRNAs begin as precursors transcribed in the nucleus and cropped
there. They move to the cytoplasm and are further cleaved by Dicer. The
RISC
with Argonaute degrades one
strand of the developing miRNA and creates the miRNA that then base
pairs with its target mRNAs in the 3' UTR. If the
base pairing is extensive the RISC causes the mRNA
to be sliced, the RISC leaves and the mRNA is rapidly degraded. If the
base pairing is less, RISC
binding causes a reduction
of translation and ultimate transfer of the mRNA to P bodies where they
are degraded.
2.
exongenous entering double-stranded RNA forms (often from viruses) are
cleaved by Dicer into shorter siRNAs. These may then be engaged by a
RISC with
Argonaute causing the destruction of one of the strands of the siRNA
which then base pairs to mRNAs, usually from the virus genes, and
proceeds to
slice them and cause their degredation as above. Alternatively, siRNAs
might engage with an RITS and Argonaute which causes the same
degredation of one strand of the
siRNA and then engages with the newly made mRNAs being made by the RNA
polymerase II to which they are
complementary. This then causes
the RITS to recruit histone modifying enzymes that work on the
chromatin structure of the genes in the transcribed
region causing transcriptional
repression.
G. Selenocysteine is
found
in a few proteins.
(not covered in 2010)
a. tRNA for selenocysteine has serine added first.
Chemically
altered to selenocysteine.
b. Interacts with a special translation factor-GTP
which
binds to the A site upstream from a signalling
stem-loop structure in the mRNA.
c. The A site has a UGA (usually a stop codon) at it
in the mRNA, but now is used as the codon for
the selenocysteine tRNA anticodon. Translation continues.
V. Control at the level of mRNA stability
A. The
3' untranslated region of eukaryotic mRNAs can contain repeated AU
sequences
that signal the poly-A tail to be rapidly degraded by nucleases.
1. This causes these mRNAs to have very short
half-lives
2. The proteins coded for by these mRNAs are usually
central to growth regulation for
the cell.
3. If a long-lived mRNA like b-globin has its 3'UTR
replaced
by these sequences
from a short-lived mRNA, it becomes short-lived also.
B.
Example
we looked at was the mRNA for the transferin receptor.
1. Has several AU repeats in its 3'UTR.
2. Allows the formation of stem-loops that contain an
iron-response element (IRE).
3. An IRE binding protein exists in two possible
conformations.
-when iron amounts are low in the cell, it is active and binds to the
IREs.
-This protects the mRNA from sigalling for degredation.
-Therefore, translation will occur and more transferin receptor will be
made.
-This will allow more iron to be brought into the cell.
-When iron levels are high, no more iron entry is needed. Aconitase
does
not bind and the mRNA
degrades.
C. We also reviewed the control
of mRNA stability at the levels of degredation from either the 5'
end, 3' end or both.