From Gene Protein – Part 1
Transcription
Lecture 16 and 17; May 26th and 31st
From Gene to Protein
DNA RNA PROTEIN
Central DogmaReplication
Transcription Translation
1940s Beadle and Tatum: One Gene – One Enzyme
Neurospora crassa (SEM)
•What is the relationship between a gene and a protein?
•Combined Biochemistry and Genetics
•Used Neurospora crassa (bread mold)
•N. crassa can live on a minimal food source
•Minimal medium (food source)•Inorganic salts•Glucose•Vitamins
Bombarded Neurospora with X-rays and then looked for mutants that differed in nutritional needs!
From Gene to Protein: Key Experiment!
1940s Beadle and Tatum: One Gene – One Enzyme
X-rays is a mutagen that causes breaks in the phosphodiester bonds of DNA which results in mutations in the DNA.
From Gene to Protein: Key Experiment!
1940s Beadle and Tatum: One Gene – One Enzyme
•Normal (wild type) neurospora can live on minimal growth medium
•Neurospora mutants need complete growth medium supplemented with all 20 amino acids
Characterized their Arginine auxotrophs
Identified 3 classes of mutants: Class I, II, and III
Prototrophs
Auxotroph
can’t live without arginine supplementation
X-rays
Wild Type
Class I
Class II
Class III
Arginine auxotrophs
From Gene to Protein: Key Experiment!
1940s Beadle and Tatum: One Gene – One Enzyme
Arginine auxotrophs 3 classes of mutants: Class I, II, and III
Arginine Biosynthesis: Ornithine and citrulline are precursors
Class IMutants
Class IIMutants
Class IIIMutantsWild type
Minimal medium(MM)(control)
MM +Ornithine
MM +Citrulline
MM +Arginine(control)
From Gene to Protein: Key Experiment!
1940s Beadle and Tatum: One Gene – One Enzyme
Arginine auxotrophs 3 classes of mutants: Class I, II, and III
Class IMutants(mutationin gene A)
Class IIMutants(mutationin gene B)
Class IIIMutants(mutationin gene C)Wild type
Gene A
Gene B
Gene C
Precursor Precursor Precursor Precursor
Ornithine Ornithine Ornithine Ornithine
Citrulline Citrulline Citrulline Citrulline
Arginine Arginine Arginine Arginine
EnzymeA
EnzymeB
EnzymeC
A A A
B B B
C C C
From Gene to Protein: Key Experiment!
From Gene to Protein: Key Experiment!
1940s Beadle and Tatum: One Gene – One Enzyme
Arginine auxotrophs 3 classes of mutants: Class I, II, and III
Precursor Ornithine Citrulline ArginineEnzyme A Enzyme B Enzyme C
Key! Different Genes involved in Arginine biosynthesis!
One Gene One Enzyme Hypothesis
One Protein
From Gene to Protein
Central Dogma
•TranscriptionIs the synthesis of RNA under the direction of DNA
Produces messenger RNA (mRNA)
•TranslationIs the actual synthesis of a polypeptide, which occurs under the direction of mRNA
Occurs on ribosomes
DNA RNA PROTEINTranscription Translation
In prokaryotes:
transcription and translation occur together
TRANSLATION
TRANSCRIPTION DNA
mRNA
Ribosome
Polypeptide
In eukaryotes:
transcription occurs in nucleus
translation occur in cytoplasm
(RNA transcripts are modified)
TRANSCRIPTION
RNA PROCESSING
TRANSLATION
mRNA
DNA
Pre-mRNA
Polypeptide
Ribosome
Nuclearenvelope
From Gene to Protein
From Gene to Protein
•Lyse a cell Remove its nuclei Collect the cytoplasm
Protein Synthesis(in vitro)
Add a Protease
Add DNase
Add RNase
No Proteins
Protein synthesis intact
Protein synthesis stopped
Message RNA was found in the 1950’s DNA RNA PROTEIN
From Gene to Protein
Dilemma:
Francis Crick: Adaptor Hypothesis
Proposed that there are 20 adaptor molecules (one for each amino acids)
Together with a set of proteins that directs the synthesis of proteins
tRNA = transfer RNAcan base pair (H-bonds) with mRNAattached to a specific amino acids
Ribosomes
How does a RNA direct the synthesis of a protein?
From Gene to Protein
Cracking the Genetic Code
How does 4 nucleotides code for 20 different amino acids?
1 nucleotide (41) only 4 amino acids
2 nucleotides (42) only 16 amino acids
3 nucleotides (43) 64 different amino acids
A triplet code is the smallest unit that can code for an amino acid!
From Gene to Protein
Cracking the Genetic Code
1961 Niremberg deciphered the first codon (triplet DNA sequence)
In a test tube (in vitro) added:
cytoplasmic extractribosomesother components (tRNA and other proteins)amino acids
Artificial mRNA:5’-UUUUUUUUU-3’5’-CCCCCCCCC-3’5’-AAAAAAAAA-3’
PHE PHE PHE PRO PRO PRO LYS LYS LYS
polypeptide
From Gene to Protein
•Genetic Code•Codon: 3 bases long (1 codon = 1 amino acid)
•64 codons total
•61 code for amino acids•More than 1 codon can encode the same amino acid
•3 STOP codons: UAG, UGA, UAA•Signals the ribosome to stop and release the protein!!
•1 Start codon: AUG encodes Methionine (MET)(every preotin starts with MET!)
From Gene to Protein
•Open Reading Frames (ORF)
ACCGCCGACUUUORF 1
THR ALA ASP PHE
PRO PRO THR
ARG ARG LEU ?
?
ORF 2
ORF 3
•Every mRNA has 3 ORF•Key! Find the start codon and you will be in the right reading frame•Ribosomes read from 5’ 3’
Genetic Code
Remember all T’s are replaced with U’s in RNA
From Gene to Protein: Transcription
•Transcription is the DNA directed synthesis of RNA
Transcriptional UNIT
P TStart Stop
Coding Region
Promoter Terminator
5’ UTR (untranslated region)Important for ribosome binding
3’ UTR (untranslated region)Important for RNA function and stability
1st base of mRNA
Note: Gene can be very big relative to the actual coding region for a given protein
5’-TATAA-3’(TATAA box Sequence- can direct synthesis from either strand)
Transcription Basics
1. Initiation• Promoter: DNA sequence that directs that start of mRNA synthesis
• RNA polymerase recognizes promoter and unwinds DNA• Promoter chooses the orientation of the gene
• Directs RNA polymerase to the right DNA strand (template)• Includes the 1st base of the mRNA
2. Elongation• RNA Polymerase synthesizes mRNA in the (5’ 3’ direction)
3. Termination• Termination sequence: stretch of DNA recognized by RNA
polymerase, which tells it to stop and release the mRNA
From Gene to Protein: Transcription
Transcription Basics: RNA synthesis
RNA polymerase – driven by PPi 2Pi (14 kCal – exergonic!)
Prokaryotes: 1 RNA polymerase
Eukaryotes: RNA Pol I“ II“ III
rRNA (ribosomal) mRNA (message) tRNA (transfer), and snRNA (small nuclear)
makes mRNA, tRNA, and rRNA
P-P-P- O
OHHO
BASEPPi
2Pi
(A,G,C,and U)
2’OH Not reactive
5’ 3’OH
RNA
3’OH reactive
From Gene to Protein: Transcription
Figure 17.7
PromoterTranscription unit
RNA polymerase
Start point
53
35
35
53
53
35
53
35
5
5
Rewound
RNA
RNA
transcript
3
3Completed RNA transcript
Unwound
DNA
RNA
transcript
Template strand of DNA
DNA
1
Initiation.
2
Elongation.
3Termination.
From Gene to Protein: Transcription
Promoter5’-TATAA-3’3’-ATATT-5’
Elongation
RNApolymerase
Non-templatestrand of DNA
RNA nucleotides
3 end
A U C C A
U
T A G G T T
AT C C A A
3
5
5
Newly madeRNA
Direction of transcription(“downstream”) Template
strand of DNA
TACTGGCGGCTGAAAGGCGGCTGA3’
ATGACCGCCGACTTTCCGCCGACT5’ 3’
5’
AUGACCGCCGACUUUCCGCCGACU5’UTR
mRNA
3’UTR
Coding region(triplet code for a.a.)
From Gene to Protein: Transcription
RNA PolymeraseUnwinds 10-20 bp at a timeMoves in the 3’5’ directionSynthesizes mRNA 5’3’
DNA is a template: chooses next base by base-paring
DNA T A
RNA A U
FidelityRNA polymerase can not proof read (check for mistakes)Codon variation helps to solve this problem
> 1 codon = 1 amino acidsError rate: 1/10,000 basesmRNA is not permanent!!
From Gene to Protein: Transcription
From Gene to Protein: Transcription
50 to 250 adenine nucleotidesadded to the 3 end
Protein-coding segment Polyadenylation signal
Poly-A tail3 UTRStop codonStart codon
5 Cap
AAUAAA AAA…AAAG P P P3
mature mRNA
5 UTR
5
A modified guanine nucleotide
added to the 5 end
5’Cap : modified Guanine - helps ribosome (in translation) find the 5’END
3’ polyAAA tail - export out of nucleus
Both 5’Cap and 3’ polyAAA tail – helps stabilize RNA in cytoplasm!!
Modification of the pre-mRNA
Eukaryotes:
From Gene to Protein: Transcription
mature mRNApre-mRNARNA processing
SPLICING
Exons: protein coding region of a gene
Introns: intervening sequence which does not code for a protein
Splicing: cut and religate mRNA in order to remove introns
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
5 CapExonIntron
1
5
30 31
Exon Intron
104 105 146
Exon 3Poly-A tail
Poly-A tail
Introns cut out andexons spliced together
Codingsegment
5 Cap1 146
3 UTR3 UTR
Pre-mRNA
mRNA
Modification of the pre-mRNAALL take place in nucleus5’Cap, 3’polyAAA tail, splicing of RNA is required to exit nucleus
Spliceosome: splices out introns to produce mature mRNA
Composed of protein and RNA
snRNA’s (small nuclear ribonucleoproteins)
base pair with intro/exon junction
cut and rejoin RNA
KEY! Spliceosome has to recognize intron/exon boundaries!!!
15% of all Inherited diseases involve splicing defects!!
From Gene to Protein: Transcription
Spliceosome
From Gene to Protein: Transcription
RNA transcript (pre-mRNA)
Exon 1 Intron Exon 2
Other proteinsProtein
snRNA
snRNPs
Spliceosome
Spliceosomecomponents
Cut-outintron
mRNA
Exon 1 Exon 2
5
5
5
1
2
3
Splicing in Eukaryotes
1. 1 gene can encode > 1 protein
2. Bigger genes: more opportunity for recombination and thus diversity!
3. Introns can regulate gene expression
From Gene to Protein: Transcription
5’Cap AAAAAAAAAAAA1 2 3 4 5
5’Cap AAAAAAAAAAAA1 2 4 5
5’Cap AAAAAAAAAAAA1 3 4 5
pre-mRNA
mature mRNA
Different tissues, different times in development, etc.
Alternative Splicing
(Prokaryotes do not modify their RNA!)
From Gene Protein – Part 2
Translation
ProkaryotesHow does the ribosome find mRNA?
Eukaryotes don’t have this! Use the 5’Cap instead
Both scan 5’3’ direction to find start codon
From Gene to Protein: Translation
P TAUG
MET (start codon)
Ribosome binding site (RBS)
CCACGCUUAA
GACACCU
*G
C* *
G U G U*
CU* G AG
GU
**A
*U
A CUC
AGACC*
C G A GA G G
G*
*GA
CUC*AU
UUAGGCG5
Amino acidattachment site
Anticodon
A3
Transfer RNA (tRNA)
Antiparallel basepairng w/ itself
Hairpin loops
Anti-codon loop that base pairs w/ mRNA codon
From Gene to Protein: Translation
3’-UAC-5’5’……….AUG…………..AAAA…3’
MET
Hydrogenbonds
Attachment of amino acid to tRNA:
61 codons but only 45 tRNAs
Wobble Theoryrelaxation of basepairing rules
5’CUU’3’5’CUA’3’5’CUG’3’5’CUC’3’
From Gene to Protein: Translation
action of aminoacyl-tRNA synthase
Leucine
Aminoacyl-tRNA synthaseBinds ATP and a.a.
Adenosine-P-P-P loses 2Piand is attached to a.a.
(14 kCal of eneergy)exergonic
Correct tRNA is bound and a.a. is transferred to tRNA.
Adenosine-monphosphate is released
Activated tRNA-amino acid molecule is released
Action of Aminoacyl-tRNA synthase!
Translation RULES
-mature mRNA-5’Cap or RBS (prokaryotes)-Ribosome scans 5’ to 3’ looking for AUG (MET) start codon-syntheses of protein in : N-term to C-term-stop codon stops synthesis
From Gene to Protein: Translation
5’…….TC ATG GAC CAT TGA G….3’3’…….AG TAC CTG GTA ACT C…..5’
DNA
Sense strand (encodes protein)
Template strand for mRNA (anti-sense)
5’…….UC AUG GAC CAU UGA G….3’mRNA
N-MET-ASP-HIS-C
Stop codon
Linked by peptide bond
Ribosomes
small – 30Slarge – 50S
A site: amino acid addition site
P site: Protein site (growing protein located here)
E site : exit site for tRNA
tRNA base pairs with the mRNA in the A and P site!
From Gene to Protein: Translation
Many proteins plus ribosomal RNA (rRNA)assembled in nucleolus
E P A
large
small
mRNA binding site
From Gene to Protein: TranslationStages of Translation
Initiationsmall subunit recognizes mRNA: 5’Cap (EUK) or RBS (PROK)scan 5’to3’ for the AUG start codonInitiator tRNAMET base pair with start codon on mRNAlarge subunit joinsother protein factors needed: “initiation factors”GTP as energy sourcetRNAMET now in the P site
Elongationgrowing peptide starts in the P siteCodon recognition
the right tRNAa.a. brought into A siteelongation factors (GTP used) needed
Formation of peptide BONDProtein now in the A siteP site has empty tRNA
TranslocationRibosome shifts 5’ to 3’
From Gene to Protein: Translation
Initiation
From Gene to Protein: Translation
Elongation
From Gene to Protein: TranslationStages of Translation – Con’t
TerminationProtein in the P siteA site has a stop codonRELEASE factor recruitedbreaks bond between peptide and tRNAstop (hydrolysis)protein is freed from ribosometRNA exits from the E siteRibosome disassembles into large and small subunits
Release
factorFree
polypeptide
Stop codon
(UAG, UAA, or UGA)
5
3 3
5
35
From Gene to Protein: TranslationPolysomes
multiple ribosomes translating protein on the same mRNAall go 5’ to 3’ direction
a way for 1 mRNA to make many proteins
From Gene to Protein: TranslationRibosomes on the Rough ER: translation of a secreted or integral
membrane protein!initiate translation the same1st 20 amino acids that exits ribosome is a signal peptiderecognized by signal-recognition particle (SRP)SRP docks ribosome onto the ER “translocation complex”SRP released and growing protein deposited into ER by ribosomeIn RER – signal peptide cleaved by proteaseElongation continuesFolding and glycosylation in RER
Figure 17.21
Ribosome
mRNA
Signal
peptide
Signal-
recognition
particle
(SRP) SRP
receptor
protein
Translocation
complex
CYTOSOL
Signal
peptide
removed
ER
membrane
Protein
ERLUMEN