Chapter 17~ From Gene to Protein
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Transcript of Chapter 17~ From Gene to Protein
Chapter 17~ From Gene to Protein
Protein Synthesis: overview
One gene-one enzyme hypothesis (Beadle and Tatum)
One gene-one polypeptide (protein) hypothesis
Transcription: synthesis of RNA under the direction of DNA (mRNA)
Translation: actual synthesis of a polypeptide under the direction of mRNA
The “Central Dogma”Flow of genetic information in a cell
How do we move information from DNA to proteins?
transcription
translation
replication
proteinRNADNA trait
DNA gets all the glory, but proteins do all the work!
mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
proteintranslation
ribosome
trait
Transcription
fromDNA nucleic acid language
toRNA nucleic acid language
RNAribose sugar N-bases
uracil instead of thymineU : AC : G
single strandedlots of RNAs
mRNA, tRNA, rRNA, siRNA…
RNADNAtranscription
TranscriptionMaking mRNA
transcribed DNA strand = template stranduntranscribed DNA strand = coding strand
same sequence as RNAsynthesis of complementary RNA strand
transcription bubbleenzyme
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
RNA polymerases3 RNA polymerase enzymes
RNA polymerase 1only transcribes rRNA genesmakes ribosomes
RNA polymerase 2transcribes genes into mRNA
RNA polymerase 3only transcribes tRNA genes
each has a specific promoter sequence it recognizes
Which gene is read?Promoter region
binding site before beginning of gene TATA box binding sitebinding site for RNA polymerase
& transcription factors
Enhancer regionbinding site far
upstream of geneturns transcription
on HIGH
Transcription FactorsInitiation complex
transcription factors bind to promoter regionsuite of proteins which bind to DNAhormones?turn on or off transcription
trigger the binding of RNA polymerase to DNA
Matching bases of DNA & RNAMatch 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
G
G
G
A
A
A
AA
5' 3'
Transcription: the process1.Initiation~
transcription factors mediate the binding of RNA polymerase to an initiation sequence (TATA box)
2.Elongation~ RNA polymerase continues unwinding DNA and adding nucleotides to the 3’ end
3.Termination~ RNA polymerase reaches terminator sequence
Eukaryotic genes have junk!Eukaryotic genes are not continuous
exons = the real geneexpressed / coding DNA
introns = the junkinbetween sequence
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
mRNA splicing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNAtranscript
mature mRNAtranscript
pre-mRNA
spliced mRNA
Post-transcriptional processing eukaryotic mRNA needs work after transcriptionprimary transcript = pre-mRNAmRNA splicing
edit out introns make mature mRNA transcript
~10,000 bases
~1,000 bases
1977 | 1993
Richard Roberts
Philip Sharp
CSHLMIT
adenovirus
common cold
Discovery of exons/introns
beta-thalassemia
Splicing must be accurateNo 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|
RNA splicing enzymes
snRNPs
exonexon intron
snRNA
5' 3'
spliceosome
exonexcisedintron
5'
5'
3'
3'
3'
lariat
exonmature mRNA
5'
No, not smurfs!“snurps”
snRNPssmall nuclear RNAproteins
Spliceosomeseveral snRNPsrecognize splice
site sequencecut & paste gene
Whoa! I think we just brokea biological “rule”!
Alternative splicingAlternative mRNAs produced from same gene
when is an intron not an intron…different segments treated as exons
Starting to gethard to define a gene!
More post-transcriptional processingNeed to protect mRNA on its trip from nucleus
to cytoplasmenzymes in cytoplasm attack mRNA
protect the ends of the moleculeadd 5 GTP capadd poly-A tail
longer tail, mRNA lasts longer: produces more protein
mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
ribosome
trait
proteintranslation
Translation
fromnucleic acid language
toamino acid language
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
AUGCGUGUAAAUGCAUGCGCCmRNA
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
codon
Cracking the code1960 | 1968
Crickdetermined 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
1960 | 1968Marshall Nirenberg
Har Khorana
The codeCode for ALL life!
strongest support for a common origin for all life
Code is redundantseveral codons for
each amino acid3rd base “wobble”
Start codon AUG methionine
Stop codons UGA, UAA,
UAG
Why is thewobble good?
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
mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
ribosome
traitaa
proteintranslation
Transfer RNA structure“Clover leaf” structure
anticodon on “clover leaf” endamino acid attached on 3 end
Loading tRNA Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNAbond requires energy
ATP AMPbond is unstableso it can release amino acid at ribosome easily
activatingenzyme
anticodontRNATrp binds to UGG codon of mRNA
Trp Trp Trp
mRNAAC CUGG
C=O
OHOH
H2OO
tRNATrp
tryptophan attached to tRNATrp
C=O
O
C=O
Ribosomes Facilitate coupling of
tRNA anticodon to mRNA codonorganelle or enzyme?
Structureribosomal RNA (rRNA) & proteins2 subunits
largesmall
E P A
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
Building a polypeptideInitiation
brings together mRNA, ribosome subunits, initiator tRNA
Elongationadding amino acids based on
codon sequence
Terminationend 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 AACCAU UG G
G AC
Val Ser
AlaTrp
releasefactor
AAA
CCU UGG 3'
Protein targeting Signal peptide
address label
Destinations: secretion nucleus mitochondria chloroplasts cell
membrane cytoplasm etc…start of a secretory pathway
Can you tell the story?
DNA
pre-mRNA
ribosome
tRNA
aminoacids
polypeptide
mature mRNA
5' GTP cap
poly-A taillarge ribosomal subunit
small ribosomal subunit
aminoacyl tRNAsynthetase
E P A
5'
3'
RNA polymerase
exon introntRNA
Prokaryote vs. Eukaryote genesProkaryotes
DNA in cytoplasmcircular
chromosomenaked DNA
no introns
EukaryotesDNA in nucleuslinear
chromosomesDNA wound on
histone proteinsintrons vs. exons
eukaryoticDNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
Transcription & translation are simultaneous in bacteria DNA is in
cytoplasmno mRNA
editing ribosomes
read mRNA as it is being transcribed
Translation in Prokaryotes
Translation: prokaryotes vs. eukaryotesDifferences between prokaryotes &
eukaryotestime & physical separation between processes
takes eukaryote ~1 hour from DNA to protein
no RNA processing
When do mutationsaffect the nextgeneration?
Mutations Point mutations
single base changebase-pair substitution
silent mutationno amino acid changeredundancy in code
missensechange amino acid
nonsensechange to stop codon
Point mutation leads to Sickle cell anemia
What kind of mutation?
Missense!
Sickle cell anemiaPrimarily Africans
recessive inheritance patternstrikes 1 out of 400 African Americans
hydrophilicamino acid
hydrophobic amino acid
Mutations Frameshift
shift in the reading framechanges everything
“downstream”insertions
adding base(s)deletions
losing base(s)
Where would this mutation cause the most change:beginning or end of gene?
Cystic fibrosisPrimarily whites of
European descentstrikes 1 in 2500 births
1 in 25 whites is a carrier (Aa)normal allele codes for a membrane protein
that transports Cl- across cell membranedefective or absent channels limit transport of Cl- (&
H2O) across cell membranethicker & stickier mucus coats around cells mucus build-up in the pancreas, lungs, digestive tract
& causes bacterial infectionswithout treatment children die before 5;
with treatment can live past their late 20s
Deletion leads to Cystic fibrosis
loss of oneamino acid
delta F508
2007-2008
What’s the value ofmutations?