Gene Expression

Gene ExpressionGene Expression

Chapter 13Chapter 13

Learning Objective 1Learning Objective 1

• What early evidence indicated that most What early evidence indicated that most genes specify the structure of proteins?genes specify the structure of proteins?

Garrod’s WorkGarrod’s Work

• Inborn errors of metabolismInborn errors of metabolism• evidence that genes specify proteins evidence that genes specify proteins

• Alkaptonuria Alkaptonuria • rare genetic disease rare genetic disease • lacks enzyme to oxidize homogentisic acidlacks enzyme to oxidize homogentisic acid

• Gene mutationGene mutation• associated with absence of specific enzymeassociated with absence of specific enzyme

AlkaptonuriaAlkaptonuria

Fig. 13-1, p. 280

Tyrosine

Functional enzyme absent

Homogentisic acid Functional enzyme present

Disease condition Normal metabolism

ALKAPTONURIA Maleylacetoacetate

Homogentisic acid excreted in urine; turns black when

exposed to air

H2OCO2

Learning Objective 2Learning Objective 2

• Describe Beadle and Tatum’s experiments Describe Beadle and Tatum’s experiments with with NeurosporaNeurospora

Beadle and TatumBeadle and Tatum

• Exposed Exposed Neurospora Neurospora sporesspores• to X-rays or ultraviolet radiationto X-rays or ultraviolet radiation• induced mutations prevented metabolic induced mutations prevented metabolic

production of essential molecules production of essential molecules

• Each mutant strainEach mutant strain• had mutation in only one genehad mutation in only one gene• each gene affected only one enzymeeach gene affected only one enzyme

Beadle-Tatum ExperimentsBeadle-Tatum Experiments

Fig. 13-2, p. 281

Expose Neurospora spores to UV light or X-rays

Fungal growth (mycelium)

Each irradiated spore is used to establish culture on complete growth medium (minimal medium plus amino acids, vitamins, etc.)

2

Transfer cells to minimal medium

plus vitamins

Transfer cells to minimal medium plus amino acids

Transfer cells to minimal medium

(control)

Minimal medium

plus arginine

Minimal medium

plus tryptophan

Minimal medium

plus lysine

Minimal medium

plus leucine

Minimal medium plus other amino

acids

1

3

KEY CONCEPTSKEY CONCEPTS

• Beadle and Tatum demonstrated the Beadle and Tatum demonstrated the relationship between genes and proteins relationship between genes and proteins in the 1940sin the 1940s

Learning Objective 3Learning Objective 3

• How does genetic information in cells flow How does genetic information in cells flow from DNA to RNA to polypeptide?from DNA to RNA to polypeptide?

DNA to ProteinDNA to Protein

• Information encoded in DNAInformation encoded in DNA• codes sequences of amino acids in proteinscodes sequences of amino acids in proteins

• 2-step process:2-step process:1. Transcription1. Transcription2. Translation2. Translation

TranscriptionTranscription

• Synthesizes Synthesizes messenger RNA (mRNA)messenger RNA (mRNA) • complementary to template DNA strandcomplementary to template DNA strand• specifies amino acid sequences of specifies amino acid sequences of

polypeptide chainspolypeptide chains

TranslationTranslation

• Synthesizes Synthesizes polypeptide chainpolypeptide chain • specified by specified by mRNAmRNA• also requires also requires tRNAtRNA and and ribosomesribosomes

• CodonCodon • sequence of 3 mRNA nucleotide basessequence of 3 mRNA nucleotide bases• specifies one specifies one amino acidamino acid • or a or a startstart or or stopstop signal signal

DNA to ProteinDNA to Protein

Fig. 13-4, p. 283

Nontemplate strand

TranscriptionDNA

Template strandmRNA

(complementary copy of

template DNA strand)

Codon 1 Codon 2 Codon 3 Codon 4 Codon 5 Codon 6

Polypeptide Met Thr Cys Glu Cys Phe

Translation

‘

‘

‘

‘

‘

‘

KEY CONCEPTSKEY CONCEPTS

• Transmission of information in cells is Transmission of information in cells is typically from DNA to RNA to polypeptidetypically from DNA to RNA to polypeptide

Learning Objective 4Learning Objective 4

• What is the difference between the What is the difference between the structures of DNA and RNA?structures of DNA and RNA?

RNARNA

• RNA nucleotidesRNA nucleotides• riboseribose (sugar) (sugar)• bases (bases (uraciluracil, adenine, guanine, or cytosine), adenine, guanine, or cytosine)• 3 phosphates3 phosphates

• RNA subunits RNA subunits • covalently joined by 5covalently joined by 5′′ – 3 – 3′′ linkages linkages• form alternating sugar-phosphate backboneform alternating sugar-phosphate backbone

RNA StructureRNA Structure

Fig. 13-3, p. 282

Uracil

Adenine

Cytosine

Guanine

Learning Objective 5Learning Objective 5

• Why is Why is genetic codegenetic code said to be redundant said to be redundant and virtually universal?and virtually universal?

• How may these features reflect its How may these features reflect its evolutionary history?evolutionary history?

Genetic CodeGenetic Code

• mRNA mRNA codonscodons• specify a sequence of amino acids specify a sequence of amino acids

• 64 codons64 codons• 61 code for amino acids61 code for amino acids• 3 codons are stop signals3 codons are stop signals

CodonsCodons

Genetic CodeGenetic Code

• Is redundantIs redundant• some amino acids have more than one codon some amino acids have more than one codon

• Is virtually universalIs virtually universal• suggesting all organisms have a common suggesting all organisms have a common

ancestorancestor• few minor exceptions to standard code found few minor exceptions to standard code found

in all organismsin all organisms

KEY CONCEPTSKEY CONCEPTS

• A sequence of DNA base triplets is A sequence of DNA base triplets is transcribed into RNA codonstranscribed into RNA codons

Learning Objective 6Learning Objective 6

• What are the similarities and differences What are the similarities and differences between the processes of between the processes of transcriptiontranscription and and DNA replicationDNA replication??

EnzymesEnzymes

• Similar enzymesSimilar enzymes• RNA polymerases RNA polymerases ((RNA synthesis)RNA synthesis)• DNA polymerases DNA polymerases ((DNA replication)DNA replication)

• Carry out synthesis in 5Carry out synthesis in 5′′ →→ 3 3′′ direction direction

• Use nucleotides with 3 phosphate groupsUse nucleotides with 3 phosphate groups

Antiparallel SynthesisAntiparallel Synthesis

• Strands of DNA are Strands of DNA are antiparallelantiparallel

• Template DNA strand and complementary Template DNA strand and complementary RNA strand are RNA strand are antiparallelantiparallel• DNA template read in 3DNA template read in 3′′ →→ 5 5′′ direction direction• RNA synthesized in 5RNA synthesized in 5′′ →→ 3 3′′ direction direction

Antiparallel SynthesisAntiparallel Synthesis

Fig. 13-9, p. 287

mRNA transcript mRNA transcriptPromoter region

Promoter region

Promoter regionRNA polymerase Gene 2

3’

Gene 1 Gene 3mRNA transcript

5’

5’

5’

3’

3’ 3’

5’

3’

5’

Base-Pairing RulesBase-Pairing Rules

• In RNA synthesis and DNA replicationIn RNA synthesis and DNA replication• are the sameare the same• exceptexcept uraciluracil is substituted for is substituted for thyminethymine

TranscriptionTranscription

Fig. 13-7, p. 286

Growing RNA strand Template DNA strand

5’ end 3’ direction

Nucleotide added to growing chain by RNA polymerase 3’end 5’ direction

Learning Objective 7Learning Objective 7

• What features of What features of tRNAtRNA are important in are important in decoding genetic information and decoding genetic information and converting it into “protein language”?converting it into “protein language”?

Transfer RNA (tRNA)Transfer RNA (tRNA)

• ““Decoding” molecule in Decoding” molecule in translationtranslation

• AnticodonAnticodon• complementary to mRNA codoncomplementary to mRNA codon• specific for 1 amino acidspecific for 1 amino acid

tRNAtRNA

Fig. 13-6a, p. 285

’ ’ Loop 3

Hydrogen bonds

Loop 1

Loop 2

Anticodon

Fig. 13-6b, p. 285

OH 3’ endAmino acid accepting end

P 5’ end

Hydrogen bonds

Loop 3Loop 1

Modified nucleotides

Loop 2

Anticodon

Fig. 13-6c, p. 285

Amino acid (phenylalanine)

Anticodon

‘ ‘

Transfer RNA (tRNA)Transfer RNA (tRNA)

• tRNAtRNA • attaches to specific amino acid attaches to specific amino acid • covalently bound bycovalently bound by aminoacyl-tRNA aminoacyl-tRNA

synthetasesynthetase enzymes enzymes

Aminoacyl-tRNAAminoacyl-tRNA

Fig. 13-11, p. 289

Phenylalanine AMP+

Aminoacyl-tRNA synthetase

+

AnticodonAmino acid tRNA Aminoacyl-tRNA

Stepped Art

Fig. 13-11, p. 289

AMP+Phenylalanine

Amino acid Aminoacyl-tRNAtRNA

+

Anticodon

Aminoacyl-tRNA synthetase

Learning Objective 8Learning Objective 8

• How do How do ribosomesribosomes function in polypeptide function in polypeptide synthesis?synthesis?

RibosomesRibosomes

• Bring together all machinery for Bring together all machinery for translationtranslation • Couple tRNAs to mRNA codonsCouple tRNAs to mRNA codons• Catalyze peptide bonds between amino acidsCatalyze peptide bonds between amino acids• Translocate mRNA to read next codonTranslocate mRNA to read next codon

Ribosomal SubunitsRibosomal Subunits

• Each ribosome is made ofEach ribosome is made of• 1 1 large ribosomal subunitlarge ribosomal subunit• 1 1 small ribosomal subunitsmall ribosomal subunit

• Each subunit containsEach subunit contains• ribosomal RNA (rRNA)ribosomal RNA (rRNA)• many proteinsmany proteins

Ribosome Ribosome StructureStructure

Fig. 13-12a, p. 290

Front view

Large subunit

E P A

Ribosome

Small subunit

Fig. 13-12b, p. 290

Large ribosomal subunit

E site

P site

A site

mRNA binding site

Small ribosomal subunit

KEY CONCEPTSKEY CONCEPTS

• A sequence of RNA codons is translated A sequence of RNA codons is translated into a sequence of amino acids in a into a sequence of amino acids in a polypeptidepolypeptide

Animation: Structure of a Animation: Structure of a RibosomeRibosome

CLICKTO PLAY

Learning Objective 9Learning Objective 9

• Describe the processes of Describe the processes of initiationinitiation, , elongationelongation, and , and terminationtermination in polypeptide in polypeptide synthesissynthesis

InitiationInitiation

• 1st stage of 1st stage of translationtranslation• Initiation factorsInitiation factors

• bind to small ribosomal subunitbind to small ribosomal subunit• which binds to mRNA at which binds to mRNA at start codon (AUG)start codon (AUG)

• Initiator tRNAInitiator tRNA• binds to start codonbinds to start codon• then binds large ribosomal subunitthen binds large ribosomal subunit

ElongationElongation

• A cyclic processA cyclic process• adds amino acids to polypeptide chainadds amino acids to polypeptide chain

• Proceeds in 5Proceeds in 5′′ →→ 3 3′′ direction along mRNA direction along mRNA

• Polypeptide chain growsPolypeptide chain grows• from amino end to carboxyl endfrom amino end to carboxyl end

TerminationTermination

• Final stage of Final stage of translationtranslation• when ribosome reaches when ribosome reaches stop codonstop codon

• AA sitesite binds to binds to release factorrelease factor• triggers release of triggers release of polypeptide chainpolypeptide chain• dissociation of translation complexdissociation of translation complex

Stages of TranscriptionStages of Transcription

RNA polymerase binds to promoter region in DNA

Termination sequence

Promoter region

Direction of transcription

RNA transcriptRewinding of DNA

Unwinding of DNA

RNA transcriptRNA polymerase

DNA

DNA

DNA template strand

Fig. 13-8, p. 287

Learning Objective 10Learning Objective 10

• What is the functional significance of the What is the functional significance of the structural differences between structural differences between bacterialbacterial and and eukaryoticeukaryotic mRNAs? mRNAs?

EukaryotesEukaryotes

• Genes Genes andand mRNA molecules mRNA molecules• are more complicated than those of bacteriaare more complicated than those of bacteria

Eukaryotic mRNAEukaryotic mRNA

• After transcriptionAfter transcription• 55′′ cap cap (modified guanosine triphosphate) is (modified guanosine triphosphate) is

added to added to 55′′ end end of mRNA molecule of mRNA molecule

• Poly-A tailPoly-A tail (adenine-containing nucleotides) (adenine-containing nucleotides)• may be added at 3may be added at 3′′ end of mRNA molecule end of mRNA molecule

Posttranscriptional ModificationPosttranscriptional Modification

Fig. 13-17, p. 295

1st exon

1st intron

2nd exon

2nd intron

3rd exon

mRNA termination sequencePromoter

Template DNA strand

7-methylguanosine cap Transcription, capping of 5’ end

5’ endStart codon Stop codon

Formation of pre-mRNASmall nuclear ribonucleoprotein complex

1st intron 2nd intron

5’ end –AAA... Poly-A tail

3’ endProcessing of pre-mRNA (addition of poly-A tail and removal of introns)

2nd exon

3rd exon

–AAA... Poly-A tail 3’ end5’ end

Protein-coding regionMature mRNA in nucleus Nuclear envelope

Nuclear pore

CytosolTransport through nuclear envelope to cytosol

–AAA... Poly-A tail 3’ end5’ end Start codon Stop codon

Mature mRNA in cytosol

1st exon

Introns and ExonsIntrons and Exons

• IntronsIntrons • noncoding regions (interrupt exons)noncoding regions (interrupt exons)• removed from original removed from original pre-mRNApre-mRNA

• ExonsExons• coding regions in eukaryotic genes coding regions in eukaryotic genes • spliced to produce continuous polypeptide spliced to produce continuous polypeptide

coding sequencecoding sequence

Learning Objective 11Learning Objective 11

• What is the difference between translation What is the difference between translation in in bacterialbacterial and and eukaryoticeukaryotic cells? cells?

Bacterial CellsBacterial Cells

• Transcription and translation are coupledTranscription and translation are coupled

• Bacterial ribosomesBacterial ribosomes• bind to 5bind to 5′′ end of growing mRNA end of growing mRNA• initiate translation before message is fully initiate translation before message is fully

synthesizedsynthesized

Bacterial mRNABacterial mRNA

Fig. 13-10, p. 288

Promoter region

mRNA termination sequenceTranscribed region

DNA

Upstream leader

sequences

Downstream trailing

sequences

Protein-coding sequences

Translated region

Start codon Stop codonmRNA

5 ′ end–OH 3 ′ end

Polypeptide

InitiationInitiation

Fig. 13-13a, p. 291

Leader sequence

mRNA

Small ribosomal subunit

Initiation factor

Start codon

Fig. 13-13b, p. 291

fMet

Initiator tRNA

Fig. 13-13c, p. 291

fMet Large ribosomal subunit

P site

E site A site

Initiation complex

ElongationElongation

Fig. 13-14, p. 292

tRNA with an amino acidAmino acids Amino acids

GDPGTP

E P A E P AAminoacyl-

tRNA binds to codon in A site

mRNA

Ribosome ready to accept another aminoacyl-tRNA

Peptide bond formation

Amino end of polypeptide New

peptide bondTranslocation

toward 3 ′ end of mRNA

E P A E P AGTP

GDP

TerminationTermination

Fig. 13-15a, p. 293

Release factor

E P A

mRNAStop codon (UAA, UAG, or UGA)

Fig. 13-15b, p. 293

Polypeptide chain is released

Stop codon (UAA, UAG, or UGA)

Fig. 13-15c, p. 293

Large ribosomal subunit

Release factor

AP

E

mRNASmall ribosomal subunit

tRNA

PolyribosomePolyribosome

• Many ribosomes bound to a single mRNAMany ribosomes bound to a single mRNA

KEY CONCEPTSKEY CONCEPTS

• Prokaryotic and eukaryotic cells differ in Prokaryotic and eukaryotic cells differ in the details of transcription and translationthe details of transcription and translation

Learning Objective 12Learning Objective 12

• Describe Describe retrovirusesretroviruses and the enzyme and the enzyme reverse transcriptasereverse transcriptase

RetrovirusesRetroviruses

• Synthesize DNA from an RNA template Synthesize DNA from an RNA template • HIV-1 (virus that causes AIDS)HIV-1 (virus that causes AIDS)

• Enzyme Enzyme reverse transcriptase reverse transcriptase • reverses flow of genetic information reverses flow of genetic information

Reverse TranscriptionReverse Transcription

Fig. 13-19a, p. 297

Chromosome DNA in nucleus of host cell Provirus inserted

into chromosome DNA

DNA provirusDNA replication

Digestion of RNA strandRNA /DNA hybrid Reverse transcription

RNA virus

Viral RNA

Fig. 13-19b, p. 297

Provirus DNA transcribed

Viral mRNA

Viral RNA

Viral proteins

RNA virus

2

Learning Objective 13Learning Objective 13

• Give examples of the different classes of Give examples of the different classes of mutations that affect the base sequence of mutations that affect the base sequence of DNA DNA

• What effects does each have on the What effects does each have on the polypeptide produced?polypeptide produced?

Base SubstitutionBase Substitution

• May alter or destroy protein function May alter or destroy protein function • missense mutationmissense mutation

• codon change specifies a different amino acidcodon change specifies a different amino acid• nonsense mutationnonsense mutation

• codon becomes a stop codoncodon becomes a stop codon

• May have minimal effectsMay have minimal effects• if amino acid is not alteredif amino acid is not altered• if codon change specifies a similar amino acidif codon change specifies a similar amino acid

Fig. 13-20a, p. 299

Normal DNA sequence

Normal mRNA sequence

Normal protein sequence

BASE-SUBSTITUTION MUTATIONS

Missense mutation

Nonsense mutation

(Stop)

(Stop)

(Stop)

Animation: Base-Pair Animation: Base-Pair SubstitutionSubstitution

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Frameshift MutationsFrameshift Mutations

• InsertionInsertion or or deletiondeletion of one or two base of one or two base pairs in a genepairs in a gene• destroys protein functiondestroys protein function• changes codon sequences downstream from changes codon sequences downstream from

the mutationthe mutation

Fig. 13-20b, p. 299

FRAMESHIFT MUTATIONS

Deletion causing nonsense

Deletion causing altered amino acid sequence

Normal DNA sequence

Normal mRNA sequence

Normal protein sequence

(Stop)

(Stop)

Animation: Frameshift Animation: Frameshift MutationMutation

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TransposonsTransposons

• Movable DNA sequencesMovable DNA sequences• ““jump” into the middle of a genejump” into the middle of a gene

• RetrotransposonsRetrotransposons• replicate by forming RNA intermediatereplicate by forming RNA intermediate• reverse transcriptase converts to original DNA reverse transcriptase converts to original DNA

sequence before jumping into genesequence before jumping into gene

KEY CONCEPTSKEY CONCEPTS

• Mutations can cause changes in Mutations can cause changes in phenotypephenotype

Animation: Protein Synthesis Animation: Protein Synthesis SummarySummary

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