Chapter 17Chapter 17From Gene to ProteinFrom Gene to Protein

Overview: The Flow of Genetic Overview: The Flow of Genetic InformationInformation

The information content of DNA is in the form of specific sequences of nucleotides

The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins

Proteins are the links between genotype and phenotype

Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation

Fig. 17-1

How does a single faulty gene result in the dramatic appearance of an albino deer?

How was the fundamental How was the fundamental relationship between genes relationship between genes and proteins discovered?and proteins discovered?

Evidence from the Study of Metabolic Evidence from the Study of Metabolic DefectsDefects

In 1909, Archibald Garrod ◦Genes dictate phenotypes through

enzymes

Symptoms of an inherited disease reflect inability to synthesize a certain enzyme

Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway

Nutritional Mutants inNutritional Mutants in Neurospora: Neurospora: Scientific InquiryScientific Inquiry

George Beadle and Edward Tatum◦ Created mutants using bread mold that

were unable to survive on minimal medium as a result of inability to synthesize certain molecules

one gene–one enzyme hypothesis◦Each gene dictates production of a specific

enzyme

Fig. 17-2a

EXPERIMENT

Growth:Wild-typecells growingand dividing

No growth:Mutant cellscannot growand divide

Minimal medium

Fig. 17-2b

RESULTSClasses of Neurospora crassa

Wild type Class I mutantsClass II mutantsClass III mutants

Minimalmedium(MM)(control)

MM +ornithine

MM +citrulline

MM +arginine(control)

Con

dit

ion

Fig. 17-2c

CONCLUSION Class I mutants(mutation in

gene A)

Class II mutants(mutation in

gene B)

Class III mutants(mutation in

gene C)Wild type

Precursor Precursor Precursor PrecursorEnzyme AEnzyme AEnzyme AEnzyme A

Ornithine Ornithine Ornithine OrnithineEnzyme BEnzyme B Enzyme BEnzyme B

Citrulline Citrulline Citrulline CitrullineEnzyme CEnzyme CEnzyme CEnzyme C

Arginine Arginine Arginine Arginine

Gene A

Gene B

Gene C

Basic Principles of Transcription & Basic Principles of Transcription & TranslationTranslation

RNA is the intermediate between genes and the proteins for which they code

Transcription is the synthesis of RNA under the direction of DNA

Transcription produces messenger RNA (mRNA)

Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA

Ribosomes are the sites of translation

• In prokaryotes, mRNA produced by transcription is immediately translated without more processing

• In a eukaryotic cell, the nuclear envelope separates transcription from translation

• Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

• A primary transcript is the initial RNA transcript from any gene

• The central dogma is the concept that cells are governed by a cellular chain of command: DNA RNA protein

Fig. 17-3a-1

TRANSCRIPTIONDNA

mRNA

(a) Bacterial cell

Fig. 17-3a-2

(a) Bacterial cell

TRANSCRIPTIONDNA

mRNA

TRANSLATIONRibosome

Polypeptide

Fig. 17-3b-1

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNA

Fig. 17-3b-2

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNARNA PROCESSING

mRNA

Fig. 17-3b-3

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNARNA PROCESSING

mRNA

TRANSLATION Ribosome

Polypeptide

The Genetic CodeThe Genetic CodeHow are the instructions for

assembling amino acids into proteins encoded into DNA?

There are 20 amino acids, but there are only four nucleotide bases in DNA

How many bases correspond to an amino acid?

Codons: Triplets of BasesCodons: Triplets of Bases

The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words

These triplets are the smallest units of uniform length that can code for all the amino acids

During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript

During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction

Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide

Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction

Each codon specifies the addition of one of 20 amino acids

Fig. 17-4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNAtemplatestrand

TRANSCRIPTION

TRANSLATION

mRNA

Protein

Codon

Amino acid

Cracking the CodeCracking the CodeAll 64 codons were deciphered by

the mid-1960sOf the 64 triplets, 61 code for amino

acids; 3 triplets are “stop” signals to end translation

The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid

Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-5Second mRNA base

Fir

st

mR

NA

base (

5 e

nd

of

cod

on

)

Th

ird

mR

NA

base (

3 e

nd

of

cod

on

)

Evolution of the Genetic CodeEvolution of the Genetic CodeThe genetic code is nearly universal,

shared by the simplest bacteria to the most complex animals

Genes can be transcribed and translated after being transplanted from one species to another

Fig. 17-6

(a) Tobacco plant expressing a firefly gene

(b) Pig expressing a jellyfish gene

TranscriptionTranscription

fromDNA nucleic acid languagetoRNA nucleic acid language

RNARNAribose sugar N-bases

◦uracil instead of thymine◦U : A◦C : G

single strandedlots of RNAs

◦ mRNA, tRNA, rRNA, siRNA…

RNADNAtranscription

3 KINDS OF RNA3 KINDS OF RNA

RIBOSOMAL RNA (rRNA)Made in nucleolus2 subunits (large & small)Combine with proteins to

form ribosomes

3 KINDS OF RNA3 KINDS OF RNA

TRANSFER RNA (tRNA)

ANTICODON sequence matches CODON on mRNA to add correctamino acids during protein synthesis

http://www-math.mit.edu/~lippert/18.417/lectures/01_Intro/

3 KINDS OF RNA3 KINDS OF RNA

MESSENGER RNA (mRNA)carries code from DNA to ribosomes

Synthesis of an RNA TranscriptSynthesis of an RNA Transcript

The three stages of transcription:◦Initiation◦Elongation◦Termination

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-7a-4Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA

2 Elongation

RewoundDNA

5

5 5 3 3 3

RNAtranscript

3 Termination

5

5

5 33

3Completed RNA transcript

TranscriptionTranscriptionMaking 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- pries the DNA strands apart

and hooks together the RNA nucleotides

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 polymerasesRNA polymerases◦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 geneTranscription factors mediate the binding of RNA

polymerase and the initiation of transcription◦ TATA box binding site◦ binding site for RNA polymerase

& transcription factors

Enhancer region◦ binding site far

upstream of gene turns transcription

on HIGH

Transcription FactorsTranscription 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

•Transcription

Matching bases of DNA & Matching bases of DNA & RNARNA

Match 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'

Eukaryotic genes have Eukaryotic genes have junk!junk!

Eukaryotic genes are not continuous◦exons = the real gene

expressed / coding DNA

◦introns = the junk inbetween sequence

eukaryotic DNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

intronscome out!

mRNA’s require EDITING before usemRNA’s require EDITING before useMessage in NOT CONTINUOUS INTRONS are removed

Image by Riedell

mRNA splicingmRNA 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 transcription◦ primary transcript = pre-mRNA◦ mRNA splicing

edit out introns ◦ make mature mRNA transcript

~10,000 bases

~1,000 bases

Splicing must be accurateSplicing 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 enzymesRNA splicing enzymessnRNPs

exonexon intronsnRNA

5' 3'

spliceosome

exonexcisedintron

5'

5'

3'

3'

3'

lariat

exonmature mRNA

5'

snRNPs◦ small nuclear RNA◦ proteins

Spliceosome◦ several snRNPs◦ recognize splice

site sequence cut & paste gene

mRNA EDITINGmRNA EDITING

ALL ENZYMES ARE PROTEINS?

RIBOZYMES-RNA molecules that function as enzymes (In some organisms pre-RNA can remove its own introns)

PROCESSING RNA

SPLICEOSOMES

Alternative splicingAlternative splicingAlternative mRNAs produced from same gene

◦ when is an intron not an intron…◦ different segments treated as exons

A A AA

A3' poly-A tail

mRNA

5'5' cap

3'

G PPP

50-250 A’s

More post-transcriptional More post-transcriptional processingprocessing

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

TranslationTranslation

fromnucleic acid languagetoamino acid language

How does mRNA code for How does mRNA code for proteins?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

mRNA codes for proteins in mRNA codes for proteins in tripletstriplets

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

Met Arg Val Asn Ala Cys Alaprotein

?

codon

The codeThe codeCode for ALL life!

◦strongest support for a common origin for all life

Code is redundant◦several codons for each amino

acid◦3rd base “wobble”- Flexible

pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon

• Start codon– AUG– methionine

• Stop codons– UGA, UAA, UAG

Why is thewobble good?

How are the codons matched How are the codons matched to amino acids?to amino acids?

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

aminoacid

tRNA anti-codon

codon

5 3

3 5

3 5

UAC

MetGCA

ArgCAU

Val

Transfer RNA structureTransfer 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

activatingenzyme

anticodontRNATrp binds to UGG

condon of mRNA

Trp Trp Trp

mRNAA C CU GG

C=O

OHOH

H2OO

tRNATrp

tryptophan attached to tRNATrp

C=O

O

C=O

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

Ribosomes 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

Protein synthesis 2

Building a polypeptideBuilding a polypeptideInitiation

◦ brings together mRNA, ribosome subunits, initiator tRNA

Elongation◦ adding amino acids based on

codon sequence

Termination◦ end 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 AACCA U UG G

G AC

ValSer

AlaTrp

releasefactor

AA A

CCU UGG 3'

How translation works

Fig. 17-18-4

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

GDPGTP

Ribosome ready fornext aminoacyl tRNA

E

P A

Protein targeting Protein targeting

Signal peptide◦address label

Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc…start of a secretory pathway

Protein Protein Synthesis in Synthesis in ProkaryotesProkaryotes

Bacterial chromosome

mRNA

Cell wall

Cellmembrane

Transcription

Psssst…no nucleus!

Prokaryote vs. Eukaryote Prokaryote vs. Eukaryote genesgenes

Prokaryotes◦ DNA in cytoplasm◦ circular

chromosome◦ naked DNA◦ no introns

Eukaryotes◦ DNA in nucleus◦ linear chromosomes◦ DNA wound on

histone proteins◦ introns vs. exons

eukaryoticDNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequenceintrons

come out!

Transcription & translation are simultaneous in bacteria ◦DNA is in

cytoplasm◦no mRNA

editing ◦ribosomes

read mRNA as it is being transcribed

Translation in ProkaryotesTranslation in Prokaryotes

Translation: prokaryotes vs. Translation: prokaryotes vs. eukaryoteseukaryotes

Differences between prokaryotes & eukaryotes◦time & physical separation between

processes takes eukaryote ~1 hour

from DNA to protein◦no RNA processing

SEE PROCESSING VIDEO

COMPLETING PROTEINSCOMPLETING PROTEINSPOLYRIBOSOMES (POLYSOMES)

◦Numerous ribosomes translate same mRNA at same time

◦3-D folding (1’, 2’, 3’ structure)◦Chaparonins

POST-TRANSLATIONAL MODIFICATIONS◦ Some amino acids modified by addition of

sugars, lipids, phosphate groups, etc◦ Enzymes can modify ends, cleave into pieces

join polypeptide strands (4’ structure)

Ex: Made as proinsulin then cut

Final insulin hormonemade of two chains connected by disulfide bridges

http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin.html

Can you tell the story?

DNA

pre-mRNA

ribosome

tRNA

aminoacids

polypeptide

mature mRNA

5' GTP cap

poly-A tail

large ribosomal subunit

small ribosomal subunit

aminoacyl tRNAsynthetase

E P A

5'

3'

RNA polymerase

exon intron

tRNA

MutationsMutationsMutation- change in genetic

material of a cell◦Point Mutations- chemical changes in

just one base pair Base-pair substitutions Base-pair insertions or deletions

Base Pair SubstitutionsBase Pair SubstitutionsBase-pair substitution-

replacement of one nucleotide and its partner, with another pair of nucleotides

silent mutation no amino acid change redundancy in code

missense Codes for different amino

acid nonsense

Codes for a stop codon

Slide from Explore Biology by Kim Foglia

Point mutation leads to Sickle cell Point mutation leads to Sickle cell anemiaanemia

What kind of mutation?

Slide from Explore Biology by Kim Foglia

Base pair substitution that caused missense mutation

Sickle cell anemiaSickle cell anemia

Slide from Explore Biology by Kim Foglia

Base Pair Insertions or Base Pair Insertions or DeletionsDeletions

Frameshift - shift in the reading frame

◦ Insertions- adding base(s)◦ Deletions- losing base(s)

Frameshift mutation- occur whenever number of nucleotides inserted or deleted are not multiples of three

changes everything “downstream”

◦ More damaging at beginning of gene than at end

Slide modified from: Explore Biology by Kim Foglia

MutagensMutagensMutagens- physical or chemical

agents that interact with DNA in ways that cause mutations◦Ex. 1920’s x-rays discovered to

cause genetic changes in fruit flies◦Application today: preliminary

screening of chemicals to identify carcinogens

WHAT IS A “GENE”?WHAT IS A “GENE”?Mendel’s factors determine phenotype

T.H. Morgan- genes located on specific chromosomes

Beadle and Tatum’s “one gene-one enzyme”

Became “One gene-one polypeptide”

- Some proteins made of more than one polypeptide chainEx: hemoglobin has 4 polypeptide chains

Now: “one gene – one polypeptide or RNA” - Not all genes code for proteins

DNA TechnologyGel

electrophoresis- technique that uses gel as a molecular sieve to separate nucleic acids or proteins based on their size, electrical charge, and other physical properties

How does it work?Restriction enzymes- enzymes that cut

DNA molecules at a limited number of specific locations◦ In nature protect cell from foreign DNA

All copies of a particular DNA molecule always yield the same set of restriction fragments when exposed to the same restriction enzyme

http://learn.genetics.utah.edu/content/labs/gel/