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BIOLOGY TEST 3
RNA SYNTHESIS IN PROKARYOTES
RNA polymerase has three requirements:
1
...

a template
3
...

Coding strand, template strand, and mRNA
holoenzyme a2BB’wo initiates RNA synthesis
core enzyme a2BB’w elongates RNA product
Sigma is only required for initiation then dissociates
...
It acts catalytically
...

Sigma decreases the affinity of the polymerase for DNA, allowing the
enzymes to rapidly scan for the promoter (consensus sequence)
...

Promoters are specific sequences that bind RNA polymerase to the initiation site
...

Closed promoter complex: promoter and the polymerase
Open promoter complex: RNA polymerase unwinds approximately 17 bases to
make DNA act as the template
...

Bubble is a DNA/RNA hybrid
...
The 3’ OH on RNA attacks the newly bound
nucleotide and forms a new phosphodiester bond, releasing pyrophosphate
...


Length of the hybrid is determined by the rudder within the polymerase
...
Hydrolysis takes
place
...

mRNA is single-stranded
form secondary structures that bind small molecules (riboswitches)
causes the RNA to become truncated, stopping translation and transcription
Summary of Prokaryotic RNA Synthesis:
1
...

initiates transcript
3
...

phosphodiester bonds are formed; RNA generated
5
...

stem loop forms/ termination
7
...
blocks elongation
Actinomycin: intercalates between the bases of the DNA double helix, preventing
the helix to be used as a template
...

mRNA comes out of polymerase
2
...

add polyA tail
all this processing happens in nucleus
export from the nuclear pore complex
translation occurs in cytoplasm
pre-mRNA contains introns and exons
mature mRNA contains only exons (coding regions)
Type - location - a amanitin effect - promotor
Polymerase 1 - nucleolus - no effect from a-amanitin - rlnr and UPE
Polymerase 2 - nucleoplasm - strongly inhibited - TATA box and enhancers
Polymerase 3 - nucleoplasm - inhibited by strong concentrations - promoters
within the genes
RNA Polymerase I - synthesize genes for rRNA - rlnr and UPE
RNA Polymerase II synthesizes protein encoding transcripts, mRNA
...

Eukaryotic promoters are called cis-acting elements: they regulate gene
elements on same molecule (e
...
TATA Box)
promoters bind to proteins called trans-acting elements of transcription factors
(e
...
TBP Tata Box Binding Protein)
Common promoters of the RNA polymerase:
1
...

initiator element (Inr) paired with the TATA Box @ start site (-3 to +5)
3
...

CAAT box ( present in things not always active; genes need to be
regulated) downstream
5
...

In eukaryotes, DNA does not interact directly with the RNA polymerase II
...

TFIIs > transcription factors recognize the promoter
Tata-Box binding protein:
TBP recognizes the TATA box
...

1
...

3
...

5
...


TFIID (with TBP) binds to TATA box
...

⁃
opens up the helix

⁃
⁃

phosphorylated the CTD
transitions into elongation

Phosphorylating the terminal forces factors to leave; only TFIIF stays
...

activators or transcription factors (trans-acting elements): bind to enhancer
(cis-acting elements) sequences on DNA
enhancers can be located 1000s of nucleotides away from start site
Mediators and DNA bending proteins act as a bridge between factors and the
RNA polymerase II
...

Examining Eukaryotic Gene expression levels:
Quantitative PCR (qPCR) or realtime PCR
1
...

2
...

DNA Microarrays
1
...

fluoroscently labelled cDNA is hybridized to reveal the expression level of
each gene
(no primer for every single one)
New genes in eukaryotic cells can be effectively expressed:
1
...

DNA is microinjected into cells
3
...

foreign mutated gene is introduced into an embryonic stem cell

2
...

4
...


recombination takes place at regions of homology
holliday junctions
normal gene is knocked out by foreign gene
knockout animals are produced

RNA Interference (RNAi) silences genes of our choice
1
...

RNA cleaved by the dicer creating siRNA
3
...

RISC unwinds one RNA strand (guide strand) bound to the RISC and
cleaves the other (passenger strand)
5
...

RNAi is used to silence any target genes
Tumor Inducing Plasmids can be used to introduce new genes into plant
cells
1
...

lump of tumor (crown gall) grows at site of infection
3
...

tumor-inducing plasmids (Ti plasmids) are carried by the A Tumerfacians
to carry instructions for the switch to the tumor state
5
...

Ti plasmid derivatives can be used as vectors to deliver foreign genes
into plant cells
...

2
...

4
...


inject active genes in baby

LECTURE 22 - RNA PROCESSING
mRNA is not modified in prokaryotes
...

1
...

nucleotides added post-transcriptionally
3
...
ALL OF THIS IS ABOUT rRNA
⁃
synthesizes 45S
⁃
45S processed to yield 18S, 28S, and 5
...

RNase P removes nucleotides from 5’ end of precursor (leader sequence)
2
...

CCA-adding enzyme adds nucleotides to the 3’ end
The sequence is removed, making the anticodon visible
...

many tRNA precursors have an intron that is removed by an endonuclease,
exposing the anticodon; translation can then occur
...

5’ end had a cap in which GTP is added with a 5’ to 5’ linkage
2
...

3’ end is cleaved by an endonuclease
4
...

introns are removed
Transcription and processing of mRNA are coupled
1
...

2)
introns spliced as synthesized

3
...


3)
4)

3’ end cleaved by an endonuclease
poly A tail added

Termination can occur before removing all introns
...

⁃
⁃
⁃
⁃
⁃

CAPPING
7-methylguanosine attached via a 5’-5’ triphosphate linkage
capping has no consensus sequence
capping stabilizes the 5’ end: protects from phosphates and nucleases
cap serves as easy recognition
all RNA Polymerase II productions have a cap

2
...

poly A tail and cap present
2
...

cleavage occurs to cut off stem-loop structure
4
...

Argonaute protein formed
silencing complexes formed
priRNA (with cap and AAA and step loop) —> Pre-mRNA (no cap and no tail) —> mature miRNA
mRNA is only one that is exported out of nucleus
...

RNA EDITING
⁃
changes proteins encoded by mRNA
⁃
codon for glutamine can be changed to a stop codon with just the
deamination of the C, making U
...

2’ OH group of branch site A attacks the 5’ splice site forms lariat
intermediate
2
...

exons join
4
...

U1 snRNP binds at 5’ splice site
2
...

U4 + U5 + U6 complex bind at intron region slpliceosome is now formed
4
...

U5 interacts with the exon sequences in the 5’ splice site and the 3’ site,
bringing both enzymes closer
6
...

U4 and U1 leave
8
...

U4 leaves; U6 uses ATP hydrolysis to displace U1 from 5’ splice site
10
...

2
...

4
...

6
...

8
...

10
...

12
...
example: calcitonin and
calcitonin-gene related peptide (CGRP)
Ribozymes function as self-splicing
1
...

Group 2 introns use A as a co-factor
3
...

1
...

C-terminal domain is phosphorylated, causing conformational change
3
...

Elongation protein complex binds to C-terminal domain of RNA
polymerase
5
...

RNA polymerase is released and dephosphorylated
7
...

Sense is the coding strand
...

the 5’ end is phosphorylated Guanine (pG)
amino acid attached to the CCA region
ester bond is formed between the animo acid and the tRNA
codon is always 5’ to 3’
anticodon is always 3’ to 5’
first position of codon is the last in the anticodon
tRNA can have INOSINE with the wobble rule

Modifications to tRNA:
1
...

dihydrouridine
3
...

First base of anticodon
C
A
U
G
I
but G)

Third base of codon
G
U
A or G
U or C
U, C, or A (everything

Aminoacyl-tRNA (charged tRNA) synthetases: catalyze the attachment of
appropriate amino acids onto the specific tRNA
⁃
forms peptide bonds
⁃
attach amino acids to the ACC arm of the tRNA
⁃
catalyzes ligation rxn w ATP
⁃
the charged amino acid is formed by an ester linkage of the carboxyl
group on the protein and the OH group on the terminal A
...

add ATP to form aminoacyl-AMP
2
...

editing site
2
...
only the serine fits
...
it keeps going until threonine is actually

put
...

error rates decrease as the protein chain length increases
...

prokaryote: 50S + 30S = 70S subunit
2
...

A - aminoacyl site binds incoming tRNA
2
...

E - exit site binds uncharged tRNA before leaving ribosome
A and P bound to the mRNA
Synthetases are true translators of the genetic code
...

Protein synthesis has three stages:
1
...

3
...
only differs in
initiation
prokaryotic mRNA is polycistronic (multiple translation start sites) one mRNA can
code for multiple proteins
...

FORMATION OF 70S INITIATION COMPLEX
1
...

IF2 and GTP - delivers fMet-tRNA to mRNA
3
...


FORMATION OF INITIATION COMPLEX REQUIRES GTP

Elongation Factors deliver aminoacyl-tRNA to the ribosome
⁃
EF-Tu and GTP deliver appropriate aminoacyl-tRNA as specified by
codon to the A site
⁃
EF-TuGDP leaves the A site prior to the peptide bond being formed
⁃
EF-Ts induce release of the GDP from EF-Tu, which is replaced with
another GTP
...

⁃
EF-Tu doesn’t interact with mRNA
⁃
EF-Tu is necessary for correct placement of aminoacyl-tRNA on
mRNA
IF11 recognizes the f-Met tRNA; EF-Tu recognize the aminoacyl-tRNAs
⁃
peptide bond catalyzed (exergonic) by a site on the 23S (component
of 50S) RNA called peptide transferase center
⁃
fMet molecule will attack the amino terminal of the amino acid in the
A site
⁃
catalytic power due to catalysis and by proximity and orientation
⁃
rRNA forms the peptide bond
⁃
Elongation Factor G (translocate) uses GTP to translocate mRNA by
one codon (binds to A site and shoves over the tRNA)
TRANSLOCATION REQUIRES GTP
Amino acid grows from N terminus to C terminus until stop codon is read
...

EFG dissociates the ribosome through GTP Hydrolysis
Two release factors in prokaryotes; one in eukaryotes
LECTURE 24
Differences b/w initiation of translation:
1
...

met-tRNA vs methionine
3
...

bacteria have two RF; eukaryotes have one
EUKARYOTES - INITIATION TRANSLATION
1
...

40S complex scans mRNA for 1st AUG using ATP
3
...

EIF-4E and EIF-4G circularize the mRNA
2
...

Prokaryotes:
Streptomycin - interferes with binding of fMet-tRNA and inhibits initiation
Tetracycline, Chloramphenicol, and Erythromycin affect elongation
Eukaryotes: Cycloheximide
Puromycin: both eukaryotes and prokaryotes
⁃
premature termination
⁃
resembles the aminoacyl terminus of amino-acyl tRNA
⁃
protein then degrades
Diphtheria toxin blocks protein synthesis in eukaryotes by inhibiting

translocation
...

Two pathways:
1
...

get half synthesized and finish in the endoplasmic reticulum
•

secretory pathway

Signal sequences mark proteins for translocation across the ER
membrane:
⁃
signal sequence is 9 - 12 hydrophobic amino acids at N-terminal
⁃
signal peptidase in the ER removes the signal
⁃
has a hydrophobic sequence, but upstream is a basic residue like
lysine K ow R Arginine
Ribosomes bound to the ER manufacture secretory membrane proteins
⁃
signal-recognition particle (SRP) uses GTP to bind signal sequence
as it exits the ribosome and directs to the ER
⁃
binding SRP to the ribosome halts protein synthesis
⁃
SRP receptor binds to the SRP ribosome complex
⁃
translocon threads the protein through the ER membrane
⁃
translation continues
⁃
signal sequence is clipped off
v-SNARE and t-SNARE direct transport vesicles to their destination in the cell
...

needs to fold (based on primary sequence)
2
...

⁃
e
...
Alzheimer’s Disease
⁃
hydrophobic patches make the protein sticky, forming aggregates
Infectious proteins called prions cause neurological diseases (folding
mistake)
⁃
exist as beta sheet rich or alpha helix rich
Molecular chaperones aid in folding BOTH USE ATP
1
...

2
...
caps it with Gro ES
...
2ATP used
...

Covalent Modifications:
1
...

Lipidation: targets proteins to membranes
3
...

Acetylation: affects charge
5
...

amidation/deamidation: specific binding tag
Proteins are subject to continuous biosynthesis and degradation
(PROTEIN TURNOVER)
⁃
endogenous proteins degrade continuously (damaged, misfiled,
unneeded)
⁃
dietary protein intake - mostly degraded
Protein turn-over:
⁃
improper folding leads to degradation
⁃
half-life of a protein is determined by the N-terminal
...

⁃
Cell cycle proteins have cyclin destruction boxes - specific amino
acid sequences that mark the protein for degradation after synthesis

⁃
other proteins have PEST sequences which also mark the protein for
quick turnover
...
E1 is not there anymore
...
only the ones that are
targeted to the proteasome are chopped up
...

helix turn helix has two alpha helices
2 binding sites
binds as a dimer
lac repressor is example of helix turn helix motif
Repressors turn gene expression off ( - ve regulation)
Activators turn gene expression on ( +ve regulation)

LAC OPERON
CAP Site: specific activator binding site to which the activator, CAP, binds
Operator: a short region of regulatory DNA to which the lac repressor
binds
lacZ: converts lactose into galactose and glucose
lacY: allows lactose to be taken up into the cell
lacA: detoxifies byproducts of the permease

ENZYME: B-galactosidase
Upstream of the lac operon is the regulator gene lacI (constitutive
expression) which has its OWN promoter
...

The lac repressor binds to operator in absence of lactose
...
(repressor falls off since
the inducer reduces its affinity for DNA) B-galactosidase makes
allolactose
...
gene transcription occurs
...

cAMP binds CAP activator
2
...

stimulates activity of RNA polymerase
Upon binding the DNA, the DNA is bent, and the RNA polymerase binds to
the promoter more effectively
...

2
...

Quorum sensing is an interaction among bacterial cells originally
discovered in bacteria living inside the bobtail squid
...

The bacteria synthesizes an enzyme called luciferase that fluorescences
the squid, protecting it
...
other cells as well as this cell both
recognize this
...

2
...

Induction: switch from lysogenic to lytic
Lytic: express all genes except repressor
Lysogenic: only repressor is expressed
Lambda repressor has highest affinity for OR1
...
repressor is bound
...

As repressor increases in concentration, it binds to second and then third
...

binding to OR1 - blocks CRO transcription
binding to OR2 - activates lambda transcription; facilitates RNA
polymerase
binding to OR3 - inhibits lambda transcription
Lysogenic: blocks expression of CRO
Lytic: chew up the repressor
...
cro expressed
...

inhibits transcription of lambda
...

when too much trp, in the leader sequence, there are two trp
...

high trp: rho-independent terminator
when a lot of trp is around
...
IT’S A SECONDARY STRUCTURE
...
will only attach when high trp levels are
there
...

Transcription:
1
...

Activation domain
GAL4 binding site
If you cut off activation site, you can bind gene, but no expression
...

THE TWO UNITS ARE MODULAR; THEY WORK INDEPENDENTLY
HOMEODOMAIN
⁃
helix turn helix
⁃
has an extra helix w interaction in the minor groove also
...

insulators: allow activators to function properly
barrier: prevent spread of heterochromatin
pluripotent: can develop into any type of fetal or adult cell
only FOUR activators are required to induce any cell to become a pluripotent
stem cell
methylation strengthens the DNA histone interactions
phosphorylation and acetylation weaken the DNA histone interactions (Facilitate
transcription)
Facilitate Transcription:
1
...

Histone kinases (HKs)
3
...

this was determined using ChIP (chromatin amino precipitation)
⁃
trapped the chromatin by formaldehyde treatment
⁃
grew them in the presence of galactose
⁃
they froze in action
⁃
chromatins were cross linked w protein
⁃
they were broken apart into pieces
⁃
Gal4 protein was attached to them
⁃
isolate the DNA from protein
⁃
sequence the DNA
There are only 10 sites out of the 4000
...

Regulation:
Methylated groups protrude into the major groove
...

An example of this would be nuclear hormone receptors
...

Estradiol binds to estrogen receptor (nuclear hormone receptor) which is
activated by binding of small molecules or ligands
...

⁃
when the ligand binds, ANOTHER PROTEIN IS RECRUITED……
It can recruit the coactivator
It can bind corepressors (upon ligand binding)
ESTROGEN RECEPTOR RECRUITS A COACTIVATOR

DRUGS
activate: agonist (estrogen receptor is example and steroids)
inhibit: antagonist (chemotherapy)
When antagonist binds, helix 12 forms a different conformation
...
genes for ferritin not made
...


EXCESS IRON
⁃
iron binds to the IRP to take it off the IRE
⁃
transferrin is not made
⁃
ferritin is made
MICRORNA miRNA
⁃
same thing as siRNA
⁃
same Araganaute protein
⁃
silence genes




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