Post on 19-Jan-2016
transcript
2007-2008 AP Biology
From Gene to Protein
How Genes Work
AP Biology
Organelles nucleus ribosomes endoplasmic reticulum
(ER) Golgi apparatus vesicles
Making proteins
smallribosomal
subunit
largeribosomal
subunit
cytoplasm
mRNA
nuclear pore
AP Biology
Nucleus & Nucleolus
AP Biology
Nucleolus Function
ribosome production build ribosome subunits from rRNA & proteins exit through nuclear pores to cytoplasm &
combine to form functional ribosomes
smallsubunit
large subunit
ribosome
rRNA &proteins
nucleolus
AP Biology
smallsubunit
largesubunitRibosomes
Function protein production
Structure rRNA & protein 2 subunits combine 0.08m
RibosomesRough
ER
SmoothER
AP Biology membrane proteins
Types of Ribosomes Free ribosomes
suspended in cytosol synthesize proteins that
function in cytosol Bound ribosomes
attached to endoplasmic reticulum
synthesize proteins for export or for membranes
AP Biology
DNA
RNA
ribosomes
endoplasmicreticulum
vesicle
Golgi apparatus
vesicle
proteinon its way!
protein finishedprotein
Making Proteins
TO:
TO:
TO:
TO:nucleus
TO:
AP Biology
TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTA
What happens in the cellwhen a gene is read?
Where are the genes?
Where does a gene start?Where does the gene end?
How do cells make proteinsfrom DNA?
How is one gene read and another one not?
How do proteinscreate phenotype?
AP Biology
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 E
disease disease disease disease
enzyme 1 enzyme 2 enzyme 3 enzyme 4
metabolic pathway
AP Biology
ingested protein
phenylalanine
tyrosine
hydroxyphenylpyruvicacid
homogentisicacid
maleylacetoaceticacid
CO2 & H2O
phenylalanine hydroxylase
transaminase
hydroxyphenylpyruvic acidoxidase
homogentisic acidoxidase
melanin
thyroxine
PKUphenylketonuria
tyrosinosis
alkaptonuria
albinism
cretinism
digestion
AP Biology
1 gene – 1 enzyme hypothesis Beadle & Tatum
Compared mutants of bread mold, Neurospora fungus created mutations by X-ray treatments
X-rays break DNA damage a gene
wild type grows on minimal media sugars + required nutrients allows fungus to
synthesize essential amino acids mutants require added amino acids
each type of mutant lacks a certain enzyme needed to produce a certain amino acid
non-functional enzyme from damaged gene
AP Biology
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 & Tatum
create mutations
positive control
negative control
experimentalsmutatio
n identifi
ed
amino acidsupplements
AP Biology
One gene / one enzyme hypothesis
chromosome
genecluster 1
enzyme E
glutamate ornithine citruline argino-succinate
arginine
enzyme F enzyme G enzyme H
encodedenzyme
substrate inbiochemical pathway
genecluster 2
genecluster 3
arg-Harg-Garg-Farg-E
Damage to specific gene, mapped to nutritional mutations
gene thatwas damaged
AP Biology
Beadle & Tatum 1941 | 1958
George Beadle
Edward Tatum
"for their discovery that genes act by regulating definite chemical events"
one gene : one enzyme hypothesis
AP Biology
The “Central Dogma” Flow of genetic information in a cell
How do we move information from DNA to proteins?
transcriptiontranslation
replication
proteinRNADNA trait
DNA gets all the glory,
but proteins do all the work!
AP Biology
RNA ribose sugar N-bases
uracil instead of thymine U : A C : G
single stranded lots of RNAs
mRNA, tRNA, rRNA, siRNA…
RNADNAtranscription
2007-2008 AP Biology
Transcription
fromDNA nucleic acid language
toRNA nucleic acid language
AP Biology
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
2007-2008 AP Biology
Transcription in Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Psssst…no nucleus!
AP Biology
Transcription in Prokaryotes Initiation
RNA polymerase binds to promoter sequence on DNA
Role of promoter Starting point
where to start reading start of gene
Template strand which strand to read
Direction on DNA always read DNA 35 build RNA 53
AP Biology
Transcription in Prokaryotes Promoter sequences
RNA polymerase molecules bound to bacterial DNA
TATAAT
RNA polymerase
Promoter
enzymesubunit
bacterial DNA
–35 sequence –10 sequenceTTGACA
RNA polymerasestrong vs. weak promoters
read DNA 35
AP Biology
Transcription in Prokaryotes
Simple proofreading 1 error/105 bases make many mRNAs mRNA has short life not worth editing!
Elongation RNA polymerase
copies DNA as it unwinds
~20 base pairs at a time 300-500 bases in gene
builds RNA 53
reads DNA 35
AP Biology
Transcription in Prokaryotes Termination
RNA polymerase stops at termination sequence
RNA GC hairpin turn
2007-2008 AP Biology
Transcription in Eukaryotes
Protein
RNA Processing
Translation
Transcription
Psssst…DNA can’t
leave nucleus!
AP Biology
Prokaryote vs. Eukaryote genes Prokaryotes
DNA in cytoplasm circular
chromosome naked DNA
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!
AP Biology
Transcription in Eukaryotes 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
AP Biology
Transcription in Eukaryotes Initiation complex
transcription factors bind to promoter region upstream of gene
suite of proteins which bind to DNA turn on or off transcription
TATA box binding site recognition site for
transcription factors
transcription factors trigger the binding of RNA polymerase to DNA
AP Biology
A A A A A3' poly-A tail
mRNA
5'5' cap
3'
G PPP
50-250 A’s
Post-transcriptional processing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNAtranscript
mature mRNAtranscript
pre-mRNA
spliced mRNA
Primary transcript (pre-mRNA) eukaryotic mRNA needs work after transcription
mRNA processing (making mature mRNA) mRNA splicing = edit out introns protect mRNA from enzymes in cytoplasm
add 5 cap add polyA tail
~10,000 bases
~1,000 bases
AP Biology
Splicing must be accurate No room for mistakes!
splicing must be exactly accurate 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|
AP Biology
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
Whoa! I think we just broke
a biological “rule”!
AP Biology
Ribozyme
Sidney Altman Thomas Cech
1982 | 1989
Yale U of Colorado
RNA as ribozyme some mRNA can even splice itself RNA as enzyme
2007-2008 AP Biology
Translation
fromnucleic acid language
toamino acid language
AP Biology
Translation Codons
blocks of 3 nucleotides decoded into the sequence of amino acids
2007-2008 AP Biology
Translation in Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Translation
proteinPsssst…no nucleus!
AP Biology
Transcription & translation are simultaneous in bacteria DNA is in
cytoplasm no mRNA
editing ribosomes
read mRNA as it is being transcribed
Translation in Prokaryotes
AP Biology
Translation: prokaryotes vs. eukaryotes Differences between prokaryotes &
eukaryotes time & physical separation between
processes takes eukaryote ~1 hour
from DNA to protein RNA processing
2007-2008 AP Biology
Translation in Eukaryotes
AP Biology
mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
mRNA leaves nucleus through nuclear pores
proteins synthesized by ribosomes using instructions on mRNA
aa
aa
aa
aa
aa
aa
aa
aa
ribosome
proteintranslation
AP Biology
How does mRNA code for proteins?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
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
AP Biology
AUGCGUGUAAAUGCAUGCGCCmRNA
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
codon
AP Biology
Cracking the code1960 | 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 (phe)
AP Biology
The 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?
AP Biology
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
AP Biology
mRNA
From gene to protein
DNAtranscription
nucleuscytoplasm
aa
aa
aa
aa
aa
aa
aa
aa
ribosome
proteintranslation
aa
AP Biology
Transfer RNA structure “Clover leaf” structure
anticodon on “clover leaf” end amino acid attached on 3 end
AP Biology
Loading tRNA Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNA bond requires energy
ATP AMP energy stored in tRNA-amino acid bond
unstable so it can release amino acid at ribosome easily
activatingenzyme
anticodontRNATrp binds to UGG
condon of mRNA
Trp Trp Trp
mRNAA C CU GG
C=O
OHOH
H2OO
tRNATrp
tryptophan attached to tRNATrp
C=O
O
C=O
AP Biology
Ribosomes Facilitate coupling of
tRNA anticodon to mRNA codon organelle or enzyme?
Structure ribosomal RNA (rRNA) & proteins 2 subunits
large small E P A
AP Biology
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
AP Biology
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 AmRNA5' 5' 5' 5'
3' 3' 3'3'
U UA AAACC
CAU UG G
GUU
A AAAC
CC
AU UG GGU
UA
AAAC
CC
AU UG GGU U
A AACCA U UG G
G AC
ValSer
AlaTrp
releasefactor
AA A
CCU UGG 3'
AP Biology
Protein targeting Signal peptide
address label
Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc…start of a secretory pathway
AP Biology
Can you tell the story?
DNA
pre-mRNA
ribosome
tRNA
aminoacids
polypeptide
mature mRNA
5' cap
polyA tail
large ribosomal subunit
small ribosomal subunit
aminoacyl tRNAsynthetase
E P A
5'
3'
RNA polymerase
exon intron
tRNA
2007-2008 AP Biology
Got Questions? Can I translate that for you?