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From Gene to Protein. How Genes Work. What do genes code for?. How does DNA code for cells & bodies? how are cells and bodies made from the instructions in DNA. DNA. proteins. cells. bodies. The “Central Dogma”. Flow of genetic information in a cell

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From Gene to Protein

How Genes Work


What do genes code for
What do genes code for?

  • How does DNA code for cells & bodies?

    • how are cells and bodies made from the instructions in DNA

DNA

proteins

cells

bodies


The central dogma
The “Central Dogma”

  • Flow of genetic information in a cell

    • How do we move information from DNA to proteins?

transcription

translation

RNA

DNA

protein

trait

DNA gets all the glory, but proteins do all the work!

replication


Metabolism taught us about genes

A

B

C

D

E

disease

disease

disease

disease

Metabolism taught us about genes

  • 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?

metabolic pathway

enzyme 1

enzyme 2

enzyme 3

enzyme 4


Beadle tatum

1941 | 1958

Beadle & Tatum

one gene : one enzyme hypothesis

George Beadle

Edward Tatum

"for their discovery that genes act by regulating definite chemical events"


X rays or ultraviolet light

Wild-type

Neurospora

asexual

spores

Minimal

medium

Growth on

complete

medium

spores

Select one of

the spores

Grow on

complete medium

Test on minimal

medium to confirm

presence of mutation

Minimal media supplemented only with…

Choline

Pyridoxine

Riboflavin

Minimal

control

Nucleic

acid

Arginine

Niacin

Inositol

Folic

acid

p-Amino

benzoic acid

Thiamine

Beadle & Tatum

create mutations

positive control

negative control

mutation identified

experimentals

amino acidsupplements


From gene to protein

aa

aa

aa

aa

aa

ribosome

aa

aa

aa

aa

aa

aa

From gene to protein

nucleus

cytoplasm

transcription

translation

DNA

mRNA

protein

trait


Transcription

Transcription

fromDNA nucleic acid languagetoRNA nucleic acid language


RNA

  • ribose sugar

  • N-bases

    • uracil instead of thymine

    • U : A

    • C : G

  • single stranded

  • lots of RNAs

    • mRNA, tRNA, rRNA, siRNA…

transcription

DNA

RNA


Transcription1
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

coding strand

3

A

G

C

A

T

C

G

T

5

A

G

A

A

A

C

G

T

T

T

T

C

A

T

C

G

A

C

T

DNA

3

C

T

G

A

A

5

T

G

G

C

A

U

C

G

U

T

C

unwinding

3

G

T

A

G

C

A

rewinding

mRNA

template strand

RNA polymerase

5

build RNA 53


Rna polymerases
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


Which gene is read
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


Transcription factors
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


Matching bases of dna rna

RNA

polymerase

Matching bases of DNA & RNA

A

  • Match RNA bases to DNA bases on one of the DNA strands

C

U

G

A

G

G

U

C

U

U

G

C

A

C

A

U

A

G

A

C

U

A

5'

3'

G

C

C

A

T

G

G

T

A

C

A

G

C

T

A

G

T

C

A

T

C

G

T

A

C

C

G

T


Eukaryotic genes have junk

intron = noncoding (inbetween) sequence

exon = coding (expressed) sequence

Eukaryotic genes have junk!

  • Eukaryotic genes are not continuous

    • exons = the real gene

      • expressed / coding DNA

    • introns = the junk

      • inbetween sequence

intronscome out!

eukaryotic DNA


Mrna splicing

intron = noncoding (inbetween) sequence

exon = coding (expressed) sequence

mRNA splicing

  • 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

eukaryotic DNA

pre-mRNA

primary mRNA

transcript

~1,000 bases

mature mRNA

transcript

spliced mRNA


Discovery of exons introns

1977 | 1993

Discovery of exons/introns

Richard Roberts

Philip Sharp

adenovirus

CSHL

MIT

common cold

beta-thalassemia


Splicing must be accurate
Splicing must be accurate

  • No room for mistakes!

    • a single base added or lost throws off the reading frame

AUGCGGCTATGGGUCCGAUAAGGGCCAU

AUGCGGUCCGAUAAGGGCCAU

AUG|CGG|UCC|GAU|AAG|GGC|CAU

Met|Arg|Ser|Asp|Lys|Gly|His

AUGCGGCTATGGGUCCGAUAAGGGCCAU

AUGCGGGUCCGAUAAGGGCCAU

AUG|CGG|GUC|CGA|UAA|GGG|CCA|U

Met|Arg|Val|Arg|STOP|


Rna splicing enzymes

snRNPs

snRNA

intron

exon

exon

5'

3'

spliceosome

5'

3'

lariat

5'

3'

exon

exon

mature mRNA

excised

intron

5'

3'

Whoa! I think

we just brokea biological “rule”!

RNA splicing enzymes

  • snRNPs

    • small nuclear RNA

    • proteins

  • Spliceosome

    • several snRNPs

    • recognize splice site sequence

      • cut & paste gene

No, not smurfs!

“snurps”


Alternative splicing
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!


More post transcriptional processing

3' poly-A tail

3'

A

A

A

A

A

mRNA

50-250 A’s

5' cap

P

P

P

5'

G

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


From gene to protein1

aa

aa

aa

aa

aa

ribosome

aa

aa

aa

aa

aa

aa

From gene to protein

nucleus

cytoplasm

transcription

translation

DNA

mRNA

protein

trait


Translation

Translation

fromnucleic acid languagetoamino acid language


How does mrna code for proteins

TACGCACATTTACGTACGCGG

DNA

AUGCGUGUAAAUGCAUGCGCC

mRNA

MetArgValAsnAlaCysAla

protein

?

How does mRNA code for proteins?

How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

4

ATCG

4

AUCG

20


Mrna codes for proteins in triplets

TACGCACATTTACGTACGCGG

DNA

AUGCGUGUAAAUGCAUGCGCC

mRNA

AUGCGUGUAAAUGCAUGCGCC

mRNA

codon

MetArgValAsnAlaCysAla

protein

?

mRNA codes for proteins in triplets


Cracking the code

1960 | 1968

Cracking the code

Nirenberg & Khorana

  • Crick

    • determined 3-letter (triplet) codon system

WHYDIDTHEREDBATEATTHEFATRAT

WHYDIDTHEREDBATEATTHEFATRAT

  • 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 | 1968

Marshall Nirenberg

Har Khorana


The code
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”

Why is thewobble good?

  • Start codon

    • AUG

    • methionine

  • Stop codons

    • UGA, UAA, UAG


How are the codons matched to amino acids

GCA

UAC

CAU

Met

Arg

Val

How are the codons matched to amino acids?

3

5

TACGCACATTTACGTACGCGG

DNA

5

3

AUGCGUGUAAAUGCAUGCGCC

mRNA

codon

3

5

tRNA

anti-codon

aminoacid


From gene to protein2

aa

aa

aa

aa

aa

ribosome

aa

aa

aa

aa

aa

aa

From gene to protein

nucleus

cytoplasm

transcription

translation

DNA

mRNA

protein

trait


Transfer rna structure
Transfer RNA structure

  • “Clover leaf” structure

    • anticodon on “clover leaf” end

    • amino acid attached on 3 end


Loading trna
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

Trp

C=O

Trp

Trp

C=O

H2O

OH

O

OH

C=O

O

activating

enzyme

tRNATrp

A

C

C

mRNA

U

G

G

anticodon

tryptophan attached to tRNATrp

tRNATrp binds to UGG condon of mRNA


Ribosomes
Ribosomes

  • Facilitate coupling of tRNA anticodon to mRNA codon

    • organelle or enzyme?

  • Structure

    • ribosomal RNA (rRNA) & proteins

    • 2 subunits

      • large

      • small

E

P

A


Ribosomes1
Ribosomes

  • 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

Met

C

A

U

5'

G

U

A

3'

E

P

A


Building a polypeptide

3

2

1

Building a polypeptide

  • Initiation

    • brings together mRNA, ribosome subunits, initiator tRNA

  • Elongation

    • adding amino acids based on codon sequence

  • Termination

    • end codon

release

factor

Leu

Val

Ser

Met

Met

Ala

Leu

Met

Met

Leu

Leu

Trp

tRNA

C

A

G

C

G

A

C

C

C

A

A

G

A

G

C

U

A

C

C

A

U

A

U

U

A

U

G

A

A

5'

5'

A

A

5'

C

U

U

5'

A

A

G

G

A

G

U

U

G

U

C

U

U

U

G

C

A

C

U

3'

G

G

U

A

A

U

A

A

C

C

mRNA

3'

3'

3'

U

G

G

U

A

A

3'

E

P

A


Protein targeting

  • Destinations:

    • secretion

    • nucleus

    • mitochondria

    • chloroplasts

    • cell membrane

    • cytoplasm

    • etc…

Protein targeting

  • Signal peptide

    • address label

start of a secretory pathway


RNA polymerase

DNA

Can you tell the story?

aminoacids

exon

intron

tRNA

pre-mRNA

5' GTP cap

mature mRNA

aminoacyl tRNAsynthetase

poly-A tail

3'

large ribosomal subunit

polypeptide

5'

tRNA

small ribosomal subunit

E

P

A

ribosome


The transcriptional unit gene

enhancer

translation

start

translation

stop

exons

1000+b

20-30b

RNA

polymerase

DNA

UTR

UTR

introns

promoter

transcription

start

transcription

stop

pre-mRNA

5'

3'

5'

3'

mature mRNA

The Transcriptional unit (gene?)

transcriptional unit (gene)

3'

5'

TAC

ACT

TATA

DNA

GTP

AAAAAAAA


Protein synthesis in prokaryotes

Bacterial chromosome

Protein Synthesis in Prokaryotes

Transcription

mRNA

Psssst…no nucleus!

Cell

membrane

Cell wall


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

intron = noncoding (inbetween) sequence

exon = coding (expressed) sequence

Prokaryote vs. Eukaryote genes

intronscome out!

eukaryotic

DNA


Translation in prokaryotes
Translation in Prokaryotes

  • Transcription & translation are simultaneous in bacteria

    • DNA is in cytoplasm

    • no mRNA editing

    • ribosomesread mRNA as it is being transcribed


Translation prokaryotes vs eukaryotes
Translation: prokaryotes vs. eukaryotes

  • Differences between prokaryotes & eukaryotes

    • time & physical separation between processes

      • takes eukaryote ~1 hour from DNA to protein

    • no RNA processing


Any Questions??

What color would a smurf turnif he held his breath?


Substitute slides for student print version

Substitute Slides for Student Print version



The transcriptional unit

enhancer

1000+b

20-30b

RNA

polymerase

5'

3'

5'

3'

The Transcriptional unit

exons

transcriptional unit

3'

5'

TAC

ACT

TATA

DNA

introns


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