RNA 10/29/10
Transcription: synthesis
Prokaryotes
- transcription initiation
o Elongation of mRNA
o Termination of transcription
- eukaryotes
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the coding part of the gene that expresses proteins through transcription and
translation (figure 1)
o can be turned on or off
o transcription initiation: (more RNA polymerase that attaches at a section)
RNA polymerase travels on 1 strand
o There are about 5000 core RNA polymerases
o 1 comes on the start, goes along, and creates RNA (elongates)
o ribosomes can translate at the same time that mRNA is begin made
Putting together the ribosome in prokaryotes: Figure 2
o Shine Dalgarno sequence: about 6 bases: where the 30s ribosome loads on for
translation, and loads and goes upstream until the AUG start codon, where a
50S is loaded
This creates a 70S full ribosome
Then, read codons to add amino acids
o In bacteria, transcription and translation happens simultaneously
Short half-life time: 3 minutes, if you stop transcription/translation
This allows bacteria to adjust to new environments quickly
Hows transcription initiated in prokaryotes? (figure 4)
o There are about 5000 core polymerases (mostly studied in e. coli gram negative
bacteria)
o TATA is about 25 base pairs in length
o Alpha alpha beta beta omega are subunits of core polymerases
o Attaches
o Sigma 70 helps locate where the TATA region is (the only transcription initiation
factor)
o After core starts to slide downstream
o CTD tails stick off alpha, attach to UP sequence
o during elongation, BPing is opening: a bubble about 14 base pairs in length of
SS DNA as polymerase copies
o Beta subunit is where Rifampcin attaches and freezes transcription in bacteria
(transcription inhibitor)
o omega is necessary to restore denatured RNA polymerase in vitro to its fully
functional form
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Methods of Termination: figure 6
o Rho independent transcription termination
the RNA snapback handle of a tennis racket is G+C rich and the 3 end of the
mRNA has 6 or >Us, hydrogen bonded to the template DNA.
As soon as base pairing happens, tennis racquet handle has G-C
Where RNA still made, U rich (about 6)
Racquet attaches to polymerase, core falls off, all ribosomes making
protein falls off the open end
Hit stop translation codon
Allow long pieces of mRNA uninterrupted, but pulling tennis racquet
causes discrete end
o Rho dependent termination
utilizes a rho factor (6 copies of a 55K Dalton monomer per rho factor) +ATP
(ADP) and the handle is less G+C rich; the 3 end of the mRNA has <Us.
Requires ATP hydrolysis
Rho protein is multi-subunit
Different because less GC rich in the handle, and doesnt have 7 Us
Rho protein attaches by polymerase,
Racquet flies up and all falls off
o
11/1/10
Eukaryotes
1. pre-mRNA by RNA polymerase II
a. general transcription factors
i. TF II: DABFEHS
ii. Enhance and double stranded DNA sequence
iii. Activator
iv. Chromatin remodeling complex
v. BRE TATA Int DCE DCE MTE DPE DCE ATG TAC
b. RNA polymerase II makes pre-mRNA
i. Travels across gene, with bubble (ss)
ii. Pre-mRNA mRNA
1. In nucleus, introns are tossed out
c. Poly-A tail
- transcription factors
o heterodimeric2 different subunits
o transcription factor II: TF II: DABFEHS
o initiation at D
o histones wrap around, bending of DNA
o chromosome remodeling complex: if transcription is turned on, then this is he tobe transcribed area of a chromosome (figure 7)
o euchromatin vs. heterochromatin: figure 8
- RNA polymerase II
o 12 subunits
o 1 has a CTD tail (largest): figure 9
CTD: carboxene terminal domain: the loading dock
One CTD tail has a set of repeating amino acids (about 52 times in
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mammals)
It hangs behind the traveling pol II downstream
Tyr ser (2) pro thr ser (5) pro ser (7)
Serine has hydroxyl group, which can be phosphorylated by kinase to
serine-phosphate (which is reversible by phosphatase)
CTD tail phosphorylation controls what is being loaded (2, 5, or 7)
If phosphorolation at these three serines, GTFs unloaded, and
transcription elongation factors TFIIs, hsPT5 loaded
de-phosphorylation unloads the elongation factors, then:
o phosphorylation at Ser 2 loads splicing factors
o phosphorylation at Ser 5 cap on 5 mRNA end. Loading of
capping factors.
o DNA proofreading (from online notes) exonucleases: XPD and XPB
o Modification of pre-mRNA as it travels
Pre-mRNA is full of E (extrons) and I (introns)
Need RNA splicing to remove introns
RNA polymerase traveling
o About 100 bases/minute
o 1. Guanylyl transferase
o 2. Methyl transferase
o if no cap, Kozac allows 40s to load next to 60s
o Transcribed from DNA RNA to make figure10b
termination of transcription and poly A
o cut loose
o add dd poly A tail
o PAP attaches puts on a few As
Poly-A-polymerase
Uses ATP
Adds 3-4 As on 3 OH end
o Then PABII
Poly-A binding protein adds 50 to 200 As
o They load via the RNA pol II CTD tail: CPSF (cleavage polyadenylation
specificity factor) and CstF (cleavage stimulation factor)
o Poly A addition is via PAP first, then PABIIp adds 50 to 200 As. These two
polymerases are loaded sequentially via the RNA pol II CTD tail
1. How does transcription start with poI/III?
a. RNA Pol I makes rRNA
b. RNA Pol III makes tRNA
c. Certain part of the chromosome codes for rRNA, not translated because not
protein
d. Figures 12/13
2. Splicing (~60s) RNA splicing
a. ~22,000 genes in human genome
b. 23 chromosomes
c. look for polymerase II coding regions
d. but more kinds of proteins because variation from not being strict with
exons/introns
e. CD40 proteinchanges in splicing programmable, metastatic (travels)
f. Small nuclear ribonuclear protein: snRNPs
i. U1 binds to 5 SS
1. + SR protein (gets U1 to bind)
a. control the binding of U1 or not
b. figure 15
2. +BBP (branch point binding)
ii. U2 binds to branch point
iii. U4, U5, U6 all attach together. U5 binds to 3 splice site
iv. U1 and U4 then off, then Lariet forms (makes 2 5 connection)
3. RNA alternative splicing
a. 4 sequences (intro) control if splicing occurs in pre-mRNA or not:
b. ESE or ISE
i. ESE SR protein, facilitates binding of U1, binds first to the exon/intron
ii. Enhances the binding of U1
c. ESS, ISS
i. Binds hnRNPs
ii. Represses U1 binding, thus splicing too
d. SR proteins (figure 15)
e. RNA editing
i. Guide RNA
ii. Deamination:
1. Ex: CAAUAA
A=UI=C (ionosine)
iii. Introns are shorter than exons
1. Splicosomes are about 60 sr from snRPs (1, 2, 3, 4, 5, 6),
machinery of a splicosome
2. Controller: SR protein family (has a long repeat of serine-argenine,
and serine can be phosphorylated)
3. Have 5 and 3 splice sites, free introns, and connect exons
f. Alternative RNA editing
i. Variations increase the types of RNA: there are 22,000 human genes, but
many more proteins found (X100), to alternatively splice original premRNAmultiply products from 1 gene
ii. SV40: infects kidney cells of African green monkeys, (the monkey virus)
105 copies of new virus from each cell infected. About 3 days
find/destroy host cell completelyliquid debris: FIGURE 16
iii. SR protein controls T or t antigen formation
iv. CD44:
1. 20 exons, 19 introns
2. mostly takes out all introns
3. exons 5, 6, 7 have variationsmetastatic cancer cells
4. not on ordinary cancel cells, tells the difference between
normal/metastatic cancer cells
5. exon 27 left outmuscular dystrophy atophy
6. melanoma
7. a few more
4. RNA editing
a. GA3 GUAUACCU
CUAACAUAUGGA mRNA
After GA3, endo cut, then UTP puts in 2 Us at the cut on top uridyl
transferase, then ligase connects
i. Consequence for ribosome: new Uschanges the set of
codonsframeshift mutation that changes the reading frames of codons
b. Deamination: CAAUAA (stop codon)
i. When C loses an amino group, it becomes U
ii. Deamination of A becomes I (Inosine)
iii. A=UI=C
1. In naturation of tRNA, made by pol III, has adenines. tRNA
converts a to I so that it binds to C instead
c. In the nucleus of a eukaryotic cell, mRNA is made in the nucleus to be exported
out
i. The 5 end 1st through a nuclear pore
ii. Proteins that escort mRNA: hnRNP, RAN protein
iii. Export signals
1. Take protein in the nucleus, signal to leave is a leucine-rich stretch
on protein about to leave (that came in). Binds to exportin 1
(another nuclear protein)
iv. Import signals
1. A protein in the cytoplasm to go to the nucleus has this sequence:
proline, lysine, lys, lys (basic), arg, lys, arg
2. Usually argine lysine rich, binds to the alpha-importin
3. Nuclear pores have many proteins associated with the double
membrane (2X PLBL)
4. Some is active transport in both directions like nTPs, DNA pol,
RNA pol is imported and exported
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translation
o A, P, E sites: Figure 18
o tRNA anticodon
aminoacyl synthases: charges the tRNA
class I: 2 OH on ribose
class II: 3 OH on ribose
charged tRNA has an amino acid
aminoacyl synthetases: takes 20 kinds of these enzymes:
1 kind hooks up a particular amino acid to connect tRNA
looks at secondary structure and places to A on 3 end correct
amino acid
adenosine has amino acids: two classes: (depending on where
you hook up the amino acid)
o class 1: 2 OH (on ribose of A)
o class II: 3 OH (on ribose of A)
anticodon on tRNA XXX
a traveling ribosome base pairs with the correct anticodon (on Asite), then hooks to carboxy end of protein (on P-site), then empty
dwells (at E site), then leaves
- initiation in prokaryotes:
* factor=protein
o 3 factors:
IF3: initiation factor 3 loads on 30s subunit to SD sequence
IF3 + 30s S.D.
30s slides to AUG, using IF2, uses GTP for energy
IF2+GTPmRNA
IF1+50s+N-formyl-met-tRNA
First codon is AUG, bring in proper tRNA to base pair with AUG,
then N-formyl removed by deformylase
1st-3rdish amino acid (sometimes) taken off
o Initiation in eukaryotes
EIF1A
eIF3
eIF5b+GTP
eIF2 + tRNA-Met
all above attach to 40s, which becomes scanning, sliding 43s , and then becomes
48s when eIF4b attaches, which activates A subunit of eIF4F to become a helicase
attaches
eIF4F (E, G, A subunits)
eIF4b
Activates A helicase (A subunit of 4F)
aminoacyl synthetases that ends correct aa to CCA ending, binds to tRNA,
and puts correct aa on CCA by looking at entire structure of tRNA
polyribosomes: many ribosomes travel in a row
eIF4F is in the cytoplasm circularizes the mRNA: figure 19
constant supply of 40s on the same strand
o Elongation in prokaryotes (of protein)
EF-Tu-GTP-tRNA chargedA site
Requires GTP hydrolysis
Bring in 1 at a time charged tRNAs into the A site
In eukaryotes, use eEF1
Peptidyl transferase
Latest amino acid is added at the COOH terminal end
Hoods amino acids together
Part of the 23s tRNA (an enzyme, not a protein)
An enzyme, not a protein
Translocation EF-G-GTP (AP site translocation)
Swinging back and forth of the polypeptide chain between the A and P
sites
Double translocation: PAP, or simple directly transport AP
Makes A site open so that when a ribosome goes downstream to next
codon, another tRNA can attach
o Termination of Translation
Ribosome falls off
STOP codons: UAA, UAG, UGA
No anticodon for tRNA, the last amino acid is whatever the codon right before
the stop codon was
In both prokaryotes and eukaryotes, the 2 subunits of the ribosome comes
apart, and the protein (COOH end) falls free
A site encounters this and no tRNA
To fall apart, release factors:
Prokaryotes:
o Pull apart the ribosome to 2 subunits: A site is open because no
amino acid, so the factor pulls in here
o RF1=UAA or UAG
o RF2 = UGA or UAA
RF3 stimulates RF1, RF2
Eukaryotes:
eRF1 + GTP to pull apart ribosome
eRF3 stimulates eRF1
- MORF
o Open reading frame: a region that can be translated by ribosomes (about 10
codons long)
o A sequence built into a region that allows correct finding of AUG
o near 5 end of message (upstream)
o a 43s eukaryotic ribosome scans looking for the correct AUG, allows 43s to
bypass incorrect AUGs
- IRES
o Internal ribosome sites: entry IRES at positions where no cap, so allows
translation anyways
If ribosome starts at an unusual place on the 5 mRNA
o You can have a start without a cap:
o 43s binds first viable protein eIF4F at a new site further downstream. eIF4F
binds and allows the 43s to scan for AUG
o Found in eukaryotic viral mRNA
- tmRNA
o piece of mRNA about 10 codons matches a tRNA and mRNA
o place where restart can happen on broken mRNA strand
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ribosome cant come off at a break, release factors cant do anything
o restart at the A site, trMNA comes in, and has a stop codon
comes tRNA, but translates its own mRNA, so ribosome can fall off, and
gives a signal for destruction of incorrectly made protein
nonsense mediated decay
o stop codon too early? No problem
o then these proteins come in: upf1, upf2, upf3 bind to stalled ribosome
o the ribosome falls off and removes the 5 cap
o 5 lytic activity (because no cap, exonuclease) destroys mRNA
nonstop mediated decay
o ribosome should stop beforehand, at the stop
o starts to translate the poly A tail on 3 eukaryotic mRNA
o decay mRNA, and protein with many lysines
o skI7 protein + exosome
sKI7 allows exosome to bind
exosome destroys mRNA (exonucleolytic)
35 decay
polylysine tail nibbled away
64 codons
o comma-less code: no space/comma between codons, its continuous
o degenerate code: 61 codons20 amino acids, for example 4 codons for valine
suppression of
o nonoverlapping: each codon is discrete (doesnt share anything with the next
base/codon)
genetic code: reading frames
o wobble allows as few as 31 kinsd of tRNA
o the code, itself: m Nirenberg experiments
o suppression of mutationsintragenic and intergenic
3 possible reading frames
o only 1 gives biologically useful (few exceptions where short mRNA sequences
read by various reading frames)
wobble
o a fast moving ribosome flys around and wobbles
o the cell can reduce the number of essential tRNA to code for amino acids
instead of 61 tRNA (how many codons there are), wobble reduces this
number to about 31
If the anticodon in the tRNA is:
The codon base can be (#3
position)
G
C, U
C
G
A
U
U
A, G
I (a base from adenine being
A, U, C
deaminated)
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o dont need as many tRNA genes, reduced exactness in A position
o in some mitochondria (which have there own DNA, xcription, xlation), 22 tRNA
UUUU
o Synthetic mRNA: if add ribosome, what protein is made?
o In vitro
o Every three codes for phenalalanine (ala)
o Shows that it has to be a triplet code (no commas)
Francis Crick, Sydney Brenner
o rII region of a T4 (a virus that infects e. coli, rII is the region)
o rII region controls how rapidly the T4 phage can lyse (destroy) the cell infected
o by using organic dyesframeshift mutations in the rII regioninsertion or
deletion of rII regiondeduce that triplet commaless code
Marshall Nirenberg
o Cracks all 61 code
20 amino acids can be separated
o can put a radioactive atom on any of the 20 amino acids (hot), and add 19 nonradioactive aa (cold)
o attach ribosome. Ribosomes a scaffold.
o Amino acid is collected onto ribosome. Any ribosome holding a cold amino acid
is not radioactive. But the 20th is hot
o Done for each amino acid separately
o Built short 3-base pair long synthetic RNA
o Goes into A site, and only stay if codon and anticodon match up
o If codon is GUG, anticodon is CAC, can match up with which amino acid was
radioactive
Examples of mutations
o Point mutation: a pase pair changes
o Insertion/deletion
o Amplification (a sequence is reiterated many times)
o Rearrangement of DNA (order changes)
Suppression of mutations
o 2nd mutation that cancels phenotypic effect of the first mutation
o intragenic
point mutation: if there is a single base change, then the amino acid
changes, and the 3-D shape of the protein is incorrect
you can have a second suppressor mutation that suppresses the bad
folding
for example, to form a dimer, the second mutaiton allows formation of a
dimer again
o intergenic
change in the anticodon in tRNA (mutation here is on another gene)
this changeit puts the correct amino acid
example: streptomycin binds the wrong bad protein drug resistance
prokaryotic operons
- operon: a set of genes that are usually between 3-12, in ds circular chromosome of
bacteria, controlled as sets. In bacterial operons, if 1 gene is on in the set, they are all
on (transcriptive) (only in bacteria)
- polycistronic mRNA: each mRNA coded by many genes in a tandem set
o example: figure 20
o coordinated set: if turn on transcription of these four genes, all at once: make a 4
gene long message (in eukaryotes, make 1 gene-length mRNA, each gene is
regulated independently)
o coordination of ratio of synthesis of these proteins
o many biocomeical pathways depend on several proteins in fixed ratios (A B
C D, each step requiring its own enzyme 1 2 or 3)
in a biological cell, need a certain amount of each converted to the next,
need to limit how much of B/C made
o cistron: gene (at RNA level)
o discovered because _______ saw genes turned on in sets learned how genes
truend on/off
o regenerative medicine (internal medicine) about transcription
o both lac and trp are polycistronic messages: figure 21
ex: Z:Y:A=100:50:30lactose operon
- Lactose (lac) operon
o Z: betagalactosidase cleaves disaccharide lactose glucose+galactose
o on/off switch for translation dependent on the concentration of lactose
o i gene has its own non-cistronic translation
o for lac repressor protein, when binds to operator site and represses ability of
mRNA to go downstream, it turns off transciption of ongoing pathway
o lactose allows discharge
if lac repressor protein binds lactose, then no represson (i genes product
cant bind to operator if lactose is present)
o high lactosemake beta-galactosidase (read polycistronic message) break
down to glucose and galactose
lactose is inducer
o the promoter is 2 parts
on right, binds RNA polymerase, but needs CAP (CRP) protein to bind for
RNA polymerase, only binds if cyclooadenosinemonophosphate (CAMP
must bind to CAP protein)
o high concentration of lactose (inducer) plus cAMP (needs low glucose)=ON
o but cant make cAMP if high concentration of glucose
o what if mutate DNA i gene? Cannot regulate transcription with lactose operon
always turned on: lactose doesnt matter but glucose does
o transcription by RNA polymerase controls how much protein is made.
Each message20-300 ribosomes
o High IPTG, a chemical that turns on the lactose operon (like lac)
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Its the natural inducer of lac operon.
Its used because it doesnt break down from beta-galactisidase,
gratuitous inducer. Causes transcription by removing the repressor
o X-gal+beta-galactisidaseblue, good color marker for the presence of betagalactisidase. Is promoter indicable by a chemical.
Tryptophan operon
o EDCBA all enzymes part of a pathway that converts chroismic acid tryptophan
o Figure 23
o Trp operon is derepressible operon
o Low concentration of trp turns on the operon (derepresses transcription)
Removes trp repressor protein from operon
So RNA polymerase can attach to TATA on promoter
(trp binds to trp repressor protein)allosteric changebinds to the
operon
o 1 is palindromic to 2, 2 is palindromic to 3, 3 is palindromic to 4
o 2 can make a tennis racquet with 3, or 3 with 4
o a tennis racquet causes attenuation (stop at 139, rho-independent termination
at 139)
o critical region number 1 has 42 bases 14 codons
2 are UGGs, 2 or trp
if not enough charged tryp, stalls at this UGG, which causes 2/3 racquet,
which goes past
This allows region 4 to be free as transcription continues (no transcription
termination at position 139) and produce an ~ 6000 base mRNA.
o how good is regulation of trp?
600 times up down gene regulation
repression: 70 fold: reduces amount of trp message when repressor
attached
attenuation: 8-10 fold: if make proteins
attenuation is only found in amino acid tpe operons
14 codons, 2 stall places??
other operons:
o Organic chemical I3P studies operon
Turns on transcription in operon
Makes sure repressor doesnt bind
Good to study rate of transctioption (how fast RNA polymerase travels)
All cells in culture represson is on
Example: when was EG message made? How soon translated?
Lambda phage virus
o Bacterial phage: a virus that infects bacteria
o A bacterial cell can attach itself: figure 24a
o Lysis produces more viruses
Lambda phage DNA replicated, and transctiption of certain genes,
translationviral proteins ot make a new virus
o Lysogeny
Integration of lambda DNA into the e. coli DNA
Homology for site on lambda circle: att site (between gal and bio) 24b
Crossing over (recombination) between lambda circle of DNA lineralized
and bacterial DNA
Att pp and BB similar crossover:
15 B.B. () are the same as pp: 24c
o need these proteins: 17:30
3 IHF protein coded by e. coli (integration host factor)
3 integrase: lambda gene protein allows crossing over
each bind to 3 sides on the pp DNA
o cos R and cos L: overhangs are homologous: easy to circularize after infetion
(24d)
o FIGURE 25
o C1 and cro code for proteins that race after infection of if cro inds to Or3 or C1
bind to Or2/Or3 (one or the other)
o Crolysis, prevents prm ( a repressor)
o C1lysogeny (prevents Pr) (a repressor)
o CIII repressor protein stabilizes CII
o Cell damage affects lysogeny
recA only binds to ss DNA: figure 26
if reduce CI, Cro takes over (then lysis)
o Xis (binds 2 sites on ds DNA) is exisionase that pulls lambda phage out
o Fis (binds 1 site) binds to ATT siteexcision of lambda DNA
o How to make a gap? Damage cells, UV light
o Recap
If binds Cro protein, lysis. Its a repressor that prevents C1 from binding
If CI protein binds, then lysogeny
RecA protein
Lysis OR lysogeny
Lysis productive infection, bacterial cell destroyed and many
phages released
Lysogeny: integration of lambda DNA to bacterial chromosome.
o recA pulls out of lysogeny because its a ss DNA binding
protein
o RecA* causes autcleavage of CI, which has a falling
concentration, so CIs destroyed, and return to lysis
At top of genetic site, OR, OR2, OR3 with promoters
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Eukaryotic gene regulation
o Protein motifs that bind to DNA: transcription factors
o DS DNA enhance + sequences like SV 40, yeast
o Chromatin remodeling
o Locus control regions ex/ globin proteins
LCR globin genes
o Signal transduction
eukaryotic gene regulation
o cell differentiation: stem? Embroyonic? How much and where transcription
regulation?
- Transcription factors are proteins that turn on genes
o Protein motif (helix 100p, leucine zipper), homeodomain protein (helix-turnhelix), zinc finger, leucine zipper, helix-loop-helix
o Has high specificity for a specific sequence in an enhancer
o WHAT Usually binds in major groove of enhancer sequence (a sequence if the
enhancer has the protein motif in its major groove, a nearby gene will be turned
on
o Example: SV40 FIGURE 28
The 72 base pair enhancer sequence: 72 72 21 21 21 Regulates level of
early or late transcription. In it, binding sites for transcription factors. Only
need one, if have one backwards, works fine. If moved somewhere else
(other site of gene) still works. Its about changing how the DNA makes
the bend.
- DS DNA enhance+sequences (eukaryote) figure 29: chromatin remodeling
- Globin: a protein in hemoglobin in RBCs
o Embreyogenesis
o 5 versions
o LCR: lows control region
o Figure
o 5 different timings, by adult, only beta version, epsilon is fetal
o figure 30 a shows the genes and b shows the regulation
- signal transduction
o signal: ligand (from outside world) binds to receptor in membrane
o signal goes across cytoplasm across into nucleus to turn on gene transctiption
o many copies of signal if similar
o enhancer sequence, all nearby genes turned on
o figure 31
o TNFalpha and IL1 is to ligandreceptortransduction
o Virus infects figure 31b