happy new year -grab a big whiteboard and a markers for your table
TRANSCRIPT
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Happy New Year
-Grab a big whiteboard and a markers for your table
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Today
• Review– DNA/RNA Structure– DNA Replication– Protein Synthesis• Transcription• mRNA processing• Translation
– Mutations
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Schedule for the week
• Tuesday – Review• Wednesday – Review for test and midterm• Thursday – Test (50 questions), review for
midterm• Friday – Review for midterm• Monday – Review for midterm• Tuesday – Friday - Midterms
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Whiteboard activity
• 1st activity– Draw a DNA molecule (3 base pairs)• Label: phosphate, deoxyribose sugar, what bases it has,
how many H bonds in between bases, 5’, 3’, purines, pyrimidines,• # the carbons of 1 sugar
– Draw a RNA molecule (3 nucleotides)• Label everything applicable
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RNA
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Whiteboard activity
• 2nd activity–Draw a replication fork• Label: helicase, SSBP’s,
toposisomerase, 5’, 3’, DNA poly III, leading strand, lagging strand, primase, primer, ligase, Okazaki fragments
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Raven - Johnson - Biology: 6th Ed. - All Rights Reserved - McGraw Hill Companies
3 options
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Question?
• After 20 rounds of DNA replication (cell division), how many cells will have parent DNA in them?
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Supercoiling
• Topoisomerases – prevent supercoiling from unraveling of DNA helix
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Telomerase – elongates
end of 5’ end of DNA to
prevent loss (end is called a telomere (TTAGGG repeat)
Small strand of RNA
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DNA Repair
• Mismatch repair: DNA polymerase
• Excision repair:Nuclease
• Telomere ends:telomerase
(may play a part in cancer treatment)
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Whiteboard activity• 3rd activity
– Protein synthesis– Write this DNA sequence3’ – TACG-CGCA-GAACC-TATGC-CCAA-GAC-TTAAA – 5’- Write the complementary mRNA- Process the mRNA
- Black segments – exons Red – introns- Put on methylated 5’ cap- Put on poly A tail
- Write the mature mRNA transcript at the bottom of the whiteboard
- Translate the mRNA by drawing the tRNA’s bonded to the mRNA and writing the corresponding amino acid it brings
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Whiteboard activity• 4th activity–Mutations
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2007-2008
From Gene to Protein
How Genes Work
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What do genes code for?
proteins cells bodies
How does DNA code for cells & bodies? how are cells and bodies made from the
instructions in DNA
DNA
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Protein Structure
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The “Central Dogma”• Flow of genetic information in a cell– How do we move information from DNA to proteins?
transcriptiontranslation
replication
protein
RNA
DNA
trait
DNA gets all the glory, but proteins do all the work!
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GENES
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Protein Synthesis
AA AA AAAA
TRANSCRIPTION TRANSLATION
DNA mRNA PROTEIN
“WRITE THEMESSAGE”
“DECODE THEMESSAGE”
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• Inheritance of metabolic diseases– suggested that genes coded for enzymes– each disease (phenotype) is caused by non-
functional gene product • lack of an enzyme• Tay sachs• PKU (phenylketonuria)• albinism
Am I just the sum of my proteins?
Metabolism taught us about genes
A B C D Edisease disease disease disease
enzyme 1 enzyme 2 enzyme 3 enzyme 4
metabolic pathway
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Beadle & Tatum 1941 | 1958
George Beadle
Edward Tatum
"for their discovery that genes act by regulating definite chemical events"
one gene : one enzyme hypothesis
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Wild-typeNeurospora
Minimalmedium
Select one ofthe spores
Grow oncomplete medium
Minimalcontrol
Nucleicacid
CholinePyridoxine Riboflavin Arginine
Minimal media supplemented only with…
ThiamineFolicacid
NiacinInositolp-Aminobenzoic acid
Test on minimalmedium to confirmpresence of mutation
Growth oncompletemedium
X rays or ultraviolet light
asexualspores
spores
Beadle & Tatumcreate mutations
positive control
negative control
experimentals mutation
identified
amino acidsupplements
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mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
aa
aa
aaaa
aa
aaaa
aa
aa
aaaa
proteintranslation
ribosome
trait
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2007-2008
Transcriptionfrom
DNA nucleic acid languageto
RNA nucleic acid language
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RNA• ribose sugar • N-bases– uracil instead of thymine– U : A– C : G
• single stranded• lots of RNAs– mRNA, tRNA, rRNA, siRNA…
RNADNAtranscription
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Transcription• Making mRNA– transcribed DNA strand = template strand– untranscribed DNA strand = coding strand
• same sequence as RNA
– synthesis of complementary RNA strand• transcription bubble
– enzyme• RNA polymerase
template strand
rewinding
mRNA RNA polymerase
unwinding
coding strand
DNAC C
C
C
C
C
C
C
C CC
G
GG
G
G G
G G
G
G
GAA
AA A
A
A
A
A
A A
A
AT
T T
T
T
T
T
T
T T
T
T
U U
5
35
3
3
5build RNA 53
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RNA polymerases• 3 RNA polymerase enzymes– RNA polymerase 1• only transcribes rRNA genes• makes ribosomes
– RNA polymerase 2• transcribes genes into mRNA
– RNA polymerase 3• only transcribes tRNA genes
– each has a specific promoter sequence it recognizes
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Which gene is read?• Promoter region– binding site before beginning of gene – TATA box binding site– binding site for RNA polymerase
& transcription factors
• Enhancer region– binding site far
upstream of gene• turns transcription
on HIGH
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Transcription Factors• Initiation complex– transcription factors bind to promoter region
• suite of proteins which bind to DNA• hormones?• turn on or off transcription
– trigger the binding of RNA polymerase to DNA
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Matching bases of DNA & RNA• Match RNA bases to DNA
bases on one of the DNA strands
U
A G GGGGGT T A C A C T T T T TC C C CA A
U
UU
U
U
G
G
A
A
A C CRNA polymerase
C
C
C
C
C
G
GG
G
A
A
A
AA
5' 3'
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Transcription video
• http://vcell.ndsu.nodak.edu/animations/home.htm
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Eukaryotic genes have junk!• Eukaryotic genes are not continuous– exons = the real gene• expressed / coding DNA
– introns = the junk• inbetween sequence
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
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mRNA splicing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNA
transcriptmature mRNA
transcript
pre-mRNA
spliced mRNA
• Post-transcriptional processing – eukaryotic mRNA needs work after transcription– primary transcript = pre-mRNA– mRNA splicing• edit out introns
– make mature mRNA transcript
~10,000 bases
~1,000 bases
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1977 | 1993
Richard Roberts Philip
SharpCSHL
MITadenovirus
common cold
Discovery of exons/introns
beta-thalassemia
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Splicing must be accurate• No room for mistakes!– a single base added or lost throws off the reading
frame
AUG|CGG|UCC|GAU|AAG|GGC|CAU
AUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
AUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGGUCCGAUAAGGGCCAU
Met|Arg|Ser|Asp|Lys|Gly|His
Met|Arg|Val|Arg|STOP|
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RNA splicing enzymes
snRNPs
exonexon intron
snRNA
5' 3'
spliceosome
exonexcisedintron
5'
5'
3'
3'
3'
lariat
exonmature mRNA
5'
No, not smurfs!“snurps”
• snRNPs– small nuclear RNA– proteins
• Spliceosome– several snRNPs– recognize splice site
sequence• cut & paste gene
Whoa! I think we just brokea biological “rule”!
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Alternative splicing• Alternative mRNAs produced from same gene– when is an intron not an intron…– different segments treated as exons
Starting to gethard to define a gene!
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A A AA
A3' poly-A tail
mRNA
5'5' cap
3'
G PPP
50-250 A’s
More post-transcriptional processing• Need to protect mRNA on its trip from nucleus
to cytoplasm– enzymes in cytoplasm attack mRNA• protect the ends of the molecule• add 5 GTP cap• add poly-A tail– longer tail, mRNA lasts longer: produces more protein
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mRNA processing video
• http://vcell.ndsu.nodak.edu/animations/home.htm
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mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
aa
aa
aaaa
aa
aaaa
aa
aa
aaaa
ribosome
trait
proteintranslation
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2007-2008
Translationfrom
nucleic acid languageto
amino acid language
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How does mRNA code for proteins?TACGCACATTTACGTACGCGG
DNA
AUGCGUGUAAAUGCAUGCGCC
mRNA
Met Arg Val Asn Ala Cys
Alaprotein
?
How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?
4
4
20
ATCG
AUCG
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AUGCGUGUAAAUGCAUGCGCC
mRNA
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGG
DNA
AUGCGUGUAAAUGCAUGCGCC
mRNA
Met Arg Val Asn Ala Cys
Alaprotein
?
codon
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Cracking the code 1960 | 1968
• Crick– determined 3-letter (triplet) codon system
Nirenberg & Khorana
WHYDIDTHEREDBATEATTHEFATRATWHYDIDTHEREDBATEATTHEFATRAT
Nirenberg (47) & Khorana (17) determined mRNA–amino acid match added fabricated mRNA to test tube of
ribosomes, tRNA & amino acids created artificial UUUUU… mRNA found that UUU coded for phenylalanine
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1960 | 1968Marshall Nirenberg
Har Khorana
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The genetic code• Code for ALL life!
– strongest support for a common origin for all life
• Code is redundant– several codons for each
amino acid– 3rd base “wobble”
Start codon AUG methionine
Stop codons UGA, UAA, UAG
Why is thewobble good?
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How are the codons matched to amino acids?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
aminoacid
tRNA anti-codon
codon
5 3
3 5
3 5
UAC
MetGCA
ArgCAU
Val
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mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
aa
aa
aaaa
aa
aaaa
aa
aa
aaaa
ribosome
traitaa
proteintranslation
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Transfer RNA structure• “Clover leaf” structure– anticodon on “clover leaf” end– amino acid attached on 3 end
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Loading tRNA • Aminoacyl tRNA synthetase – enzyme which bonds amino acid to tRNA– bond requires energy
• ATP AMP• bond is unstable• so it can release amino acid at ribosome easily
activatingenzyme
anticodontRNATrp binds to UGG condon of mRNA
Trp Trp Trp
mRNAA C CU G G
C=OOH
OHH2OO
tRNATrp
tryptophan attached to tRNATrp
C=O
O
C=O
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Ribosomes
• Facilitate coupling of tRNA anticodon to mRNA codon– organelle or enzyme?
• Structure– ribosomal RNA (rRNA) & proteins– 2 subunits• large• small E P A
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Ribosomes
Met
5'
3'
UUA C
A G
APE
• A site (aminoacyl-tRNA site) – holds tRNA carrying next amino acid to be
added to chain • P site (peptidyl-tRNA site) – holds tRNA carrying growing polypeptide chain
• E site (exit site)– empty tRNA
leaves ribosome from exit site
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Building a polypeptide• Initiation
– brings together mRNA, ribosome subunits, initiator tRNA
• Elongation– adding amino acids based on codon
sequence
• Termination– end codon 123
Leu
Leu Leu Leu
tRNA
Met MetMet Met
PE AmRNA
5' 5' 5' 5'3' 3' 3' 3'
U UA A AACC
CA U UG G
GUU
A AAAC
CC
A U UG GGU
UA A A
ACC
CA U UG G
GU UA A ACCA U UG G
G A C
Val Ser
Ala Trp
releasefactor
AA A
C CU UG G 3'
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Translation video
• http://vcell.ndsu.nodak.edu/animations/home.htm
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Protein targeting • Signal peptide– address label
Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc…start of a secretory pathway
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Can you tell the story?
DNA
pre-mRNA
ribosome
tRNA
aminoacids
polypeptide
mature mRNA
5' GTP cap
poly-A tail
large ribosomal subunit
small ribosomal subunit
aminoacyl tRNAsynthetase
E P A
5'
3'
RNA polymerase
exon introntRNA
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AAAAAAAAGTP
20-30b
3'
promoter transcriptionstop
transcriptionstart
introns
The Transcriptional unit (gene?)
transcriptional unit (gene)TAC ACT
DNA
DNATATA5'RNA
polymerase
pre-mRNA5' 3'
translationstart
translationstop
mature mRNA5' 3'
UTR UTR
exonsenhancer 1000+b
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2007-2008
Protein Synthesis in Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Psssst…no nucleus!
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Prokaryote vs. Eukaryote genes• Prokaryotes– DNA in cytoplasm– circular
chromosome– naked DNA – no
proteins– no introns
• Eukaryotes– DNA in nucleus– linear
chromosomes– DNA wound on
histone proteins– introns vs. exons
eukaryoticDNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
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• Transcription & translation are simultaneous in bacteria – DNA is in
cytoplasm– no mRNA
editing – ribosomes
read mRNA as it is being transcribed
Translation in Prokaryotes
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Translation: prokaryotes vs. eukaryotes• Differences between prokaryotes &
eukaryotes– time & physical separation between processes• takes eukaryote ~1 hour
from DNA to protein– no RNA processing
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2007-2008
Any Questions??What color would a smurf turnif he held his breath?