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?