Sean Carroll
http://www.molbio.wisc.edu/carroll/teaching.html
sbcarrol@wisc.edu
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Suggestions for doing well
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Come to class !
Read the assignment before lecture
Stay current with problems
Seek help if needed
You are responsible for content of lectures
and readings (even if not in lecture notes)
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Where are we heading?
From DNA
To Diversity
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How are we going to get there?
• Understanding how genetic information is
encoded and decoded - DNA, RNA, proteins and
the genetic code
• Understanding how the expression of genetic
information is regulated in simpler and more
complex organisms
• Understanding how evolutionary changes in
protein function and expression arise and shape
organismal diversity
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Molecular Genetics Overview
I.
Rules of the Gene
DNA
RNA
Protein
(The Central Dogma)
II.
Gene Regulation in Prokaryotes and Eukaryotes
How and Why Gene Expression is Controlled in Biological
Processes
III.
Development and Evolution of Animals
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“The Central Dogma”
Lectures 14-18
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Requirements of the Genetic Material
• Information -
must direct the organization
and metabolic activities of
the cell
• Replication must replicate accurately so
that the information it
contains is inherited by
daughter cells
• Mutation must undergo occasional
mutation, so that new heritable
phenotypes arise
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Genes Are Made of DNA
Historical Developments
1870 - Miescher shows DNA is in the nucleus
1912 - Feulgen shows:
- specific DNA stain patterns in nucleus
- quantity of stain in nucleus fixed in a species
- following meiosis amount of stain reduced by 1/2
- during mitotic interphase amount of stain 2X
1942 - UV light causes mutations
260nm is best wavelength
260nm is wavelength DNA absorbs
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Griffith’s discovery of transformation in
Streptococcus pneumoniae (1928)
© 2003 John Wiley and Sons Publishers
9
Avery, MacLeod, and McCarty’s proof that
the “transforming principle” is DNA.
© 2003 John Wiley and Sons Publishers
10
Demonstration by Hershey and Chase that the
genetic information of bacteriophage T2 resides in its DNA.
© 2003 John Wiley and Sons Publishers
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Key Structural Features of DNA
1.
Double Helical
due to bond angles, the helix twists such that one turn is
made about every 10bp
2.
Antiparallel - Polarity
5’
3’ on one strand
3’
5’ on the other
3.
Base Pairing - Complementarity
via H bonds , C G A = T (Four Bases)
Chargaff’s Rule %C equals %G, %A equals %T
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Diagram of a DNA double helix.
© 2003 John Wiley and Sons Publishers
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Diagram of a DNA double helix.
Structures of the Four DNA nucleotides
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RNA
DNA
Strandedness
Single
(usually)
Double
Sugar
Ribose
(2’ - OH)
Deoxyribose
(2’ - H)
Uracil
Thymine
Base
composition
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Fig. 10.12 Diagram of a DNA double helix.
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H bonds are only about 3% as strong as covalent bonds. Why would this be important?
Recommended reading- much better than
any textbook you will ever use
The Eighth Day of Creation:
The Makers of the Revolution in
Biology
Horace Freeland Judson
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Replication
Purpose: Propagate copies with high fidelity
Problem: How does one copy a doublestranded template?
e.g.
Read intact?
1. Read both
strands
2. Conserve
parental
sequence
Alternatives:
1.
Semi-conservative - Daughters segregate with one parental strand
2.
Conservative - Daughters segregate together
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black = new strand
How to distinguish these two possibilities?
The Messelson-Stahl Experiment
"The Most Beautiful Experiment in Biology"
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Meselson and Stahl’s experiment demonstrating that DNA
replicates by a semiconservative mechanism in E. coli.
1. Label all DNA with "Heavy"
isotope of nitrogen 15N, the
density will be heavier than
normal 14N DNA.
2. Transfer growing cells to 14N
media, after different
generations, extract DNA and
centrifuge in CsCl gradient
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DNA Replication: Enzymology
Enzymes catalyze the formation and destruction of chemical
bonds
There is a direction and chemistry to the replication of DNA
REQUIREMENTS
1. Deoxynucleotide triphosphates (dNTP) provide energy
and monomer units of DNA.
2. A single stranded template must be present. DNA will
not polymerize without a template.
3. A primer must be present to initiate chain synthesis.
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Mechanism of action of DNA polymerase I:
covalent extension of a DNA primer strand in the 5’  3’ direction.
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Replication in vivo is Bi-Directional from Origins
"Theta Structures"
Unit of Replication = Replicon
E. coli has one = one origin of replication
Eukaryotes have multiple origins
Rate = 1000 nucleotides / second
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Anatomy of a Replication Fork
Discontinuous replication (the
lagging strand)
Okazaki fragments
Direction of fork
Continuous replication (leading strand)
1. Synthesis is only in 5' 3' direction
2. Must use primer
(RNA made by primase & dnaB).
3. Exonuclease edits in 3' 5' direction
(KEY)
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Synthesis of the lagging strand
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Enzymology : A Bacterial “Replisome”
1. Unwinds
4. Synthesizes RNA primer
2. Protects ssDNA
3. DNA polymerase III
5' -> 3' chain elongation
5. Fills gaps
6. Ligates fragments
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Who Cares ?
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A Eukaryotic Problem : “Split Ends”
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The Solution - Telomerase
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The Solution - Telomerase
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Who Cares ?
• Most somatic cells make little or no
telomerase so chromosomes become shorter
and cells senesce
• Cancer cells make telomerase and thus
remain mitotic - telomerase is thus
chemotherapy target
• Defects in telomerase machinery associated
with premature aging
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”Central Dogma"
Replication
DNA
RNA
(Transcription)
(Translation)
Protein
(Translation)ription)
TRANSCRIPTION:
Process of synthsizing RNA from a DNA template.
Unlike replication, where all DNA is copied,
transcription is selective, only certain regions of the
DNA are transcribed and these are, in general, GENES.
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Credit: From D.
Prescott, “Cellular
Sites of RNA
Synthesis, “ Prog.
Nucleic Acid Res.
Mol. Biol. 3:33-57,
1964.
Autoradiographs demonstrating the synthesis of RNA in the
nucleus and its subsequent transport to the cytoplasm.
© 2003 John Wiley and Sons Publishers
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Credit: From D.
Prescott, “Cellular
Sites of RNA
Synthesis, “ Prog.
Nucleic Acid Res.
Mol. Biol. 3:33-57,
1964.
Autoradiographs demonstrating the synthesis of RNA in the
nucleus and its subsequent transport to the cytoplasm.
© 2003 John Wiley and Sons Publishers
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Transcription
3 Basic Types of RNA molecules (others
later), each with different functions
1. mRNA messenger
RNA
2. tRNA
transfer RNA
3. rRNA
ribosomal
RNA
Carries information to
ribosome where it is
translated
Involved in decoding the
mRNA into the actual
protein
Part of the ribosome
23S RNA in 50S subunit
16S RNA in 30S subunit
5S RNA in 50S subunit
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Features of Transcription
1. Complementarity
The RNA is complementary to one strand of the DNA
2. Initiation
RNA polymerase initiates at specific sites (PROMOTERS)
3. Termination
There are specific signals to stop transcription
RNA is single stranded
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Complementarity
Transcription
DNA
3' - AATCCGCCTAT - 5'
5' - TTAGGCGGATA - 3'
transcription is 5'
3'
transcript is complementary to DNA, then
RNA
5' - UUAGGCGGAUA - 3'
Note that uracil is used in place of thymine
RNA
DNA
Single stranded
Uses ribose
(ribonucleotides)
Uracil, A, G, C
Double stranded
Uses deoxyribose
(deoxyribonucleotides)
Thymine, A, G, C
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RNA polymerase The enzyme that synthesizes RNA polymers from DNA template
- uses ATP, UTP, CTP, GTP (not dNTPs)
- must recognize the beginning and end of genes
- it does so by recognizing signature sequences:
In PROMOTERS - the initiation sequences is
at the 5' end of genes (e.g. the lac genes of E. coli)
GENERAL IMPORTANCE: The decision to transcribe a gene or not is
often regulated at initiation and mediated by physiological controls.
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RNA Polymerase Recognizes Specific
Sequences in Promoters that then
Position the Start of Transcription
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Sigma Factor Recognizes and Binds to
the Promoter
The “Core” Enzyme Synthesizes the RNA transcript
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Transcription in Genomes
Asymmetrical
Can be either strand of DNA
3'
5'
5'
3'
In Eukaryotes
Transcription is in the nucleus but RNA is transported to the cytoplasm
Highly Regulated
Growth, development, response to environment all require selective
expression of genes in different cell types or in different environments
(more, much, more, later)
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Comparing transcription and RNA
processing in prokaryotes and eukaryotes
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Key differences in eukaryotic
gene structure and expression
1. Introns
2. RNA splicing
3. polyadenylation
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© 2003 John Wiley and Sons Publishers
KNOW THE IMPORTANT
DIFFERENCES IN GENE
STRUCTURE AND RNA
PROCESSING BETWEEN
PROKARYOTES AND
EUKARYOTES,
but you are not responsible for
biochemical details of differences in
transcriptional machinery, or how RNA
is spliced or poly adenylated
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Review and understand
RNA synthesis utilizes only one DNA strand of a gene as template.
© 2003 John Wiley and Sons Publishers
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"CENTRAL DOGMA"
Replication
DNA
RNA
(Transcription)
(Translation)
Protein
(Translation)ription)
TRANSLATION:
Process of synthsizing protein from an RNA template.
Usually, one continuous stretch of the RNA sequence is
decoded into protein (the reading frame)
Lots of Components to Translation Machinery
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Proteins
• Polymers of amino acids
• Average 300-500 amino acids in length
• 20 different amino acids in all
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R is one of 20 structures
Fig 13.1 Structures of the 20 amino acids commonly found in proteins.
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A protein forms by polymerization
Polymerization is brought about by coding and
decoding RNA molecules, by enzymes, by energy,
all in a macromolecular assembly complex.
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The Genetic Code
How does the sequence of nucleotide pairs dictate the sequence of amino
acids in the protein?
i.e. what is the CODE?
• Is the code overlapping or non-overlapping?
• How many bases make up an amino acid?
• Which bases encode which amino acids?
LOGIC: Re # of Bases / Amino Acid
Not 1,
Not 2,
3?,
there's 4 bases and 20 Amino Acids
42 = 16
43 = 64 possible triplets
Maybe it's 2 and they're overlapping?
- Nope, mutants show only single changes
Sort it Out Genetically
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Genetics of rII Rapid lysis Gene of T4
(a bacteriophage)
“Solving the Code without Opening the Black Box’
Strategy:
Create Suppressors of rII mutations using Proflavin
which inserts or deletes single bases
Starting with a mutant, then adding or deleting a nucleotide to see which will
restore Reading Frame
+1
+1
+1
-1
+1
+2
restores
does not
restores
e.g.
ABC ABC ABC ABC
+1
AAB CAB CAB CAB
frame shifted
+2
AAA BCA BCA BCA
frame shifted
+3
AAA ABC ABC ABC
RESTORED
Reading frame is a triplet
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Features of the Code
1.
Degeneracy
64 triplets but 20 amino acids
If there were only 20 triplets used, then 44 triplets would be
nonsense as would most mutations. But, since most frame-shifts
still make a protein, then most triplets are used. Therefore,
individual amino acids are encoded by >1 triplet.
2.
Non-overlapping
3.
Triplet Code
4.
Sequence is read from a fixed beginning point to the end of
coding sequence. Mutations which shift frame alter sequence all
the way through to the end.
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Cracking the Code - Biochemically
Synthetic RNA, polymerized in a test tube
E. coli translation goop
(ribosomes, amino acyl tRNAs, energy, enzymes)
Protein
Experiment 1
Make poly-U (UUUUU…), add goop
Result: Poly phenylalanine
UUU encodes phynylalanine
CCC encodes proline
AAA encodes lysine
GGG encodes glycine
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Experiment 2
Mix bases in different ratios, look at ratios of amino acids
produced
Probability of Codon = Fraction of Amino Acid
e.g.
Mix U:G at 3:1 ratio
Probability of UUU is (3/4)3 = 27/64
3:1
Probability of UUG is (3/4)(3/4)(1/4) = 9/64
Ratio of phenylalanine: leucine = 3:1
UUG encodes leucine
We couldn't always tell which sequence encoded amino acid, just the
composition
Experiment 3
• Use defined synthetic trinucleotides
• See which radioactive aminoacyl tRNAs are bound to ribosomes
Whole code decipherable
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The Genetic Code
64 possible codons, 61 used.
Some amino acids use >1 codon e.g. GGX = glycine
3 codons = STOP
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The adapter: tRNA
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tRNA - Amino Acid Relationships
1. Certain amino acids can come to the ribosome via
different tRNAs.
2. Certain tRNAs can bring their amino acids in
response to several codons - through a loose binding
property of anticodon called WOBBLE
There are only about 50 tRNAs in an E. coli cell. The
third position of anticodon is not as constrained.
Thus, tRNA
3'
5'
UAI 5' will pair with:
AUA 3'
AUC
all isoleucine
AUA
I = inosine, a modified base
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Wobble Rules
5' end of anticodon
G
C
A
U
I
3' end of codon
U or C
G only
U only
A or G
U, C, or A
From viewpoint
of tRNA
anticodon
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Base pairing between the anticodon of alanyl-tRNAAla1 and
mRNA codons GCU, GCC, and GCA according to Crick’s
wobble hypothesis.
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Nonsense Suppressors
Mutations in anticodon loop of tRNA can allow recognition
of stop codon by aminoacyl tRNA.
e.g.
tyr
tyr
3' AUG 5'
3' AUC 5'
tyr
3'
5'
AUC
UAG
5'
Stop codon becomes
tyr codon
3'
Other tyr tRNAs put tyr in right places.
Other stop codons get used downstream.
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Information Flow
DNA
3'
5'
5'
3'
Transcription
mRNA
5'
3'
tRNA anticodon
Translation
Protein
N
C
But what about the chemistry of protein synthesis,
what are roles of mRNA, tRNA, rRNA, and
ribosome?
Approach it as a stepwise process.
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A snapshot of translation in progress
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Stepwise:
1. Attachment of Amino Acids to tRNA
3'
3'
tRNA "cloverleaf" structure
• All tRNAs have A at the 3' end
3'
5'
5'
5'
•Amino acid is attached to 3' OH via
carboxyl group via a tRNA synthetase
aa1 + tRNA1 + GTP
aa1 - tRNA1 + GDP
Anticodon
"charged" tRNA
Generally: Same number of amino acyl tRNA synthetases as amino acids.
BUT, not the same number of tRNAs as amino acids
Question: At site of protein synthesis, which gets
recognized, the tRNA or the amino acid?
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Nifty Experiment:
Take cysteinyl tRNA (tRNAcys) and treat with nickel hydride, which alters
cysteine to alanine - what happens to protein?
Ala
Cys
5'
3'
3'
5'
5'
+ Ni
Anticodon
Anticodon
Result: Everywhere one would expect Cys, you get Ala.
Amino acids are illiterate, the tRNA recognizes the
mRNA sequence, not the amino acid.
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Step 2: Formation of the Initiation Complex
Every protein begins with methionine (f-met E. coli)
In E. coli: One tRNA encodes f-met, one encodes reg. Met
The mRNA is read 3 bases at a time. The codon is a TRIPLET.
The met codon is
5’ AUG 3’
the tRNA anticodon. 3’ UAC 5’
3’
MET
5’
IN THE RIBOSOME:
A series of complexes are formed.
3’ UAC 5’
mRNA
5’ AUG 3’
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The First Formation is
mRNA + 30S ribosome subunit +IF3 (initiation factor 3)
(IF3 keeps 30S subunit dissociated from 50S subunit, IF1 and
IF2 ensure that only the initiator tRNA enters the P site)
The second formation is
GTP + tRNAfmet + IF2
IF2 forms complex with tRNA
NOTE: ONLY DURING INITIATION
DOES tRNA ENTER VIA P SITE
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(Recycle
IF1 and
IF2)
The third formation is the 70S
initiation complex
30S subunit contains Decoding Center
50S subunit contains peptidyl
transferase center
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Step 3: Elongation
I. Second tRNA is positioned at the A site hydrogen bonded via its
anticodon to the next codon on the mRNA
Elongation Factors:
>EF-Tu ~ GTP
For binding and positioning of tRNA at A site
II. Peptide Bond Formation
P
mRNA
tRNA1
A
tRNA2
Peptide Bond
P site depleted,
A site has peptide
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II. ELONGATION
Note that tRNAs now have 5’
anticodon and 3’ end in
different sites in the large and
small subunits - they must next
move the anticodon ends to the
corresponding sites in the
small subunit 69
III. Translocation
EF-G fits into A site (of large subunit)
Moves A
P site
Moves P
E site
mRNA moves three bases, exposing new codon
-
Creates new A site for next tRNA
tRNA in E site leaves
IV. Termination
Three mRNA codons are nonsense or STOP codons
UAG UAA UGA
When these are found in the A site, release factors
block further elongation.
mRNA is released, ribosome dissociates and protein
is free
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III. TRANSLOCATION
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IV. TERMINATION
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In the Ribosome:
1.
Τhe 5’ end of the mRNA usually is complementary to short stretch of
16SrRNA which helps to position initiator tRNA in P site
2. The P site is the site of the growing chain
The A site is the site of the new tRNA
The E site is the site of tRNA exit
(P-Peptidyl, A-Aminoacyl E -Exit - THINK ABOUT IT)
3.
The 50S subunit associates with the initiation complex
4.
20 peptide bonds/second
5.
90% of energy in E. coli goes for protein synthesis.
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DNA - RNA - PROTEIN
Information Flow - Derive One Sequence From Another
DNA
3’
TAC
ATA
GTA
CTA
CCC
ACG
ATC
5’
RNA
5’
AUG
UAU
CAU
GAU
GGG
UGC
UAB
3’
GLY -
CYS -
PROTEIN
MET - TYR -
HIS - ASP -
GIVEN RNA OR DNA SEQUENCE, ALL CAN BE DEDUCED
GIVEN PROTEIN SEQUENCES, THERE ARE AMBIGUITIES
e.g.
ASP
Possible
mRNA
GAU
GAC
GAU
C
5’
mRNA
DNA
GAC
U
3’
CTG
A
-
ARG
CGX
AGA
AGG
X
CGG
A A
X
TCC
G T
-
VAL
GUX
-
TYR -
ILE
UAU
UAC
AUU
AUC
AUA
GUX
UAU
C
CAX
ATA
G
C
AUU
A
G
TAA
T
-
HIS
CAU
CAC
-
PRO
CCX
3’
CAU
C
CCX
GTA
G
GGX
5’
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SOME BIOLOGICAL FACTS TO KNOW:
In Eukaryotes:
• transcription in nucleus
• translation in cytoplasm
(uncoupled)
• all genes monocistronic
• 5’ cap on mRNA
In Prokaryotes:
• no nucleus
• translation can begin
before transcription is
finished
• Some genes are
polycistronic
• rRNA sequence
ΑΝΤΙΒΙΟΤΙCS
Puromycin - binds A site, terminates translation
Streptomycin, Tetracycline, chloramphenicol all inhibit various aspects of translation
ORGANELLES
Mitochondria & chloroplasts have own genomes that probably evolved from
prokaryotes - These organelles’ protein synthesis is sensitive to prokaryotic antibiotics
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