from gene protein part 1 transcription - class...

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!


X-rays is a mutagen that causes breaks in the phosphodiester bonds of DNA which results in mutations in the DNA.



•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



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)




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





Precursor Ornithine Citrulline ArginineEnzyme A Enzyme B Enzyme C

Key! Different Genes involved in Arginine biosynthesis!

One Gene One Enzyme Hypothesis

One 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



•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


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?


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!


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


•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!)


•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


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


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.


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.)


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!!



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:


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!!


Spliceosome


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


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


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’


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


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!


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’


Initiation


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

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