Gene Expression

1
Gene Expression

• Chapter 13

2
Learning Objective 1

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

3
Garrods Work

• Inborn errors of metabolism
• evidence that genes specify proteins
• Alkaptonuria
• rare genetic disease
• lacks enzyme to oxidize homogentisic acid
• Gene mutation
• associated with absence of specific enzyme

4
Alkaptonuria
5

Tyrosine
Homogentisic acid
Functional enzyme absent
Functional enzyme present
Disease condition
Normal metabolism
ALKAPTONURIA
Maleylacetoacetate
Homogentisic acid excreted in urine turns black
when exposed to air
H2O
CO2
Fig. 13-1, p. 280
6
Learning Objective 2

• Describe Beadle and Tatums experiments with
  Neurospora

7
Beadle and Tatum

• Exposed Neurospora spores
• to X-rays or ultraviolet radiation
• induced mutations prevented metabolic production
  of essential molecules
• Each mutant strain
• had mutation in only one gene
• each gene affected only one enzyme

8
Beadle-Tatum Experiments
9

Expose Neurospora spores to UV light or X-rays
1
Each irradiated spore is used to establish
culture on complete growth medium (minimal medium
plus amino acids, vitamins, etc.)
Fungal growth (mycelium)
2
Transfer cells to minimal medium plus amino acids
Transfer cells to minimal medium plus vitamins
Transfer cells to minimal medium (control)
3
Minimal medium plus arginine
Minimal medium plus tryptophan
Minimal medium plus lysine
Minimal medium plus leucine
Minimal medium plus other amino acids
Fig. 13-2, p. 281
10
KEY CONCEPTS

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

11
Learning Objective 3

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

12
DNA to Protein

• Information encoded in DNA
• codes sequences of amino acids in proteins
• 2-step process
• 1. Transcription
• 2. Translation

13
Transcription

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

14
Translation

• Synthesizes polypeptide chain
• specified by mRNA
• also requires tRNA and ribosomes
• Codon
• sequence of 3 mRNA nucleotide bases
• specifies one amino acid
• or a start or stop signal

15
DNA to Protein
16

Nontemplate strand



Transcription
DNA


mRNA (complementary copy of template DNA strand)
Template strand

Codon 1
Codon 2
Codon 3
Codon 4
Codon 5
Codon 6
Polypeptide
Met
Thr
Cys
Glu
Cys
Phe
Translation
Fig. 13-4, p. 283
17
KEY CONCEPTS

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

18
Learning Objective 4

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

19
RNA

• RNA nucleotides
• ribose (sugar)
• bases (uracil, adenine, guanine, or cytosine)
• 3 phosphates
• RNA subunits
• covalently joined by 5' 3' linkages
• form alternating sugar-phosphate backbone

20
RNA Structure
21

Uracil
Adenine
Cytosine
Guanine
Fig. 13-3, p. 282
22
Learning Objective 5

• Why is genetic code said to be redundant and
  virtually universal?
• How may these features reflect its evolutionary
  history?

23
Genetic Code

• mRNA codons
• specify a sequence of amino acids
• 64 codons
• 61 code for amino acids
• 3 codons are stop signals

24
Codons
25
Genetic Code

• Is redundant
• some amino acids have more than one codon
• Is virtually universal
• suggesting all organisms have a common ancestor
• few minor exceptions to standard code found in
  all organisms

26
KEY CONCEPTS

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

27
Learning Objective 6

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

28
Enzymes

• Similar enzymes
• RNA polymerases (RNA synthesis)
• DNA polymerases (DNA replication)
• Carry out synthesis in 5' ? 3' direction
• Use nucleotides with 3 phosphate groups

29
Antiparallel Synthesis

• Strands of DNA are antiparallel
• Template DNA strand and complementary RNA strand
  are antiparallel
• DNA template read in 3' ? 5' direction
• RNA synthesized in 5' ? 3' direction

30
Antiparallel Synthesis
31

mRNA transcript
mRNA transcript
Promoter region
Promoter region
Promoter region
5
5
Gene 2
RNA polymerase
5
5
3
3
3
3
3
Gene 1
Gene 3
5
mRNA transcript
Fig. 13-9, p. 287
32
Base-Pairing Rules

• In RNA synthesis and DNA replication
• are the same
• except uracil is substituted for thymine

33
Transcription
34

Growing RNA strand
Template
DNA strand
5 end
3 direction
Nucleotide added to growing chain by RNA
polymerase
5 direction
3end
Fig. 13-7, p. 286
35
Learning Objective 7

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

36
Transfer RNA (tRNA)

• Decoding molecule in translation
• Anticodon
• complementary to mRNA codon
• specific for 1 amino acid

37
tRNA
38


Loop 3

Hydrogen bonds
Loop 1
Loop 2
Anticodon
Fig. 13-6a, p. 285
39

OH 3 end
Amino acid accepting end
P 5 end
Hydrogen bonds
Loop 3
Loop 1
Modified nucleotides
Loop 2
Anticodon
Fig. 13-6b, p. 285
40
Amino acid (phenylalanine)


Anticodon
Fig. 13-6c, p. 285
41
Transfer RNA (tRNA)

• tRNA
• attaches to specific amino acid
• covalently bound by aminoacyl-tRNA synthetase
  enzymes

42
Aminoacyl-tRNA
43

AMP
Phenylalanine

Aminoacyl-tRNA synthetase
Anticodon
Amino acid
tRNA
Aminoacyl-tRNA
Fig. 13-11, p. 289
44

Stepped Art
Fig. 13-11, p. 289
45
Learning Objective 8

• How do ribosomes function in polypeptide
  synthesis?

46
Ribosomes

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

47
Ribosomal Subunits

• Each ribosome is made of
• 1 large ribosomal subunit
• 1 small ribosomal subunit
• Each subunit contains
• ribosomal RNA (rRNA)
• many proteins

48
Ribosome Structure
49

Front view
Large subunit
E P A
Ribosome
Small subunit
Fig. 13-12a, p. 290
50

Large ribosomal subunit
E site
P site
A site
Small ribosomal subunit
mRNA binding site
Fig. 13-12b, p. 290
51
KEY CONCEPTS

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

52
Animation Structure of a Ribosome
CLICKTO PLAY
53
Learning Objective 9

• Describe the processes of initiation, elongation,
  and termination in polypeptide synthesis

54
Initiation

• 1st stage of translation
• Initiation factors
• bind to small ribosomal subunit
• which binds to mRNA at start codon (AUG)
• Initiator tRNA
• binds to start codon
• then binds large ribosomal subunit

55
Elongation

• A cyclic process
• adds amino acids to polypeptide chain
• Proceeds in 5' ? 3' direction along mRNA
• Polypeptide chain grows
• from amino end to carboxyl end

56
Termination

• Final stage of translation
• when ribosome reaches stop codon
• A site binds to release factor
• triggers release of polypeptide chain
• dissociation of translation complex

57
Stages of Transcription
58
RNA polymerase binds to promoter region in DNA
DNA
Termination sequence
Promoter region
Direction of transcription
DNA template strand
RNA transcript
Rewinding of DNA
Unwinding of DNA
DNA
RNA transcript
RNA polymerase
Fig. 13-8, p. 287
59
Learning Objective 10

• What is the functional significance of the
  structural differences between bacterial and
  eukaryotic mRNAs?

60
Eukaryotes

• Genes and mRNA molecules
• are more complicated than those of bacteria

61
Eukaryotic mRNA

• After transcription
• 5' cap (modified guanosine triphosphate) is added
  to 5' end of mRNA molecule
• Poly-A tail (adenine-containing nucleotides)
• may be added at 3' end of mRNA molecule

62
Posttranscriptional Modification
63

mRNA termination sequence
1st exon
1st intron
2nd exon
2nd intron
3rd exon
Promoter
Template DNA strand
Transcription, capping of 5 end
7-methylguanosine cap
5 end
Start codon
Stop codon
Formation of pre-mRNA
1st intron 2nd intron
Small nuclear ribonucleoprotein complex
5 end
AAA... Poly-A tail 3 end
Processing of pre-mRNA (addition of poly-A tail
and removal of introns)
2nd exon
3rd exon
1st exon
AAA... Poly-A tail 3 end
5 end
Protein-coding region
Mature mRNA in nucleus
Nuclear envelope
Nuclear pore
Cytosol
Transport through nuclear envelope to cytosol
AAA... Poly-A tail 3 end
5 end
Stop codon
Start codon
Mature mRNA in cytosol
Fig. 13-17, p. 295
64
Introns and Exons

• Introns
• noncoding regions (interrupt exons)
• removed from original pre-mRNA
• Exons
• coding regions in eukaryotic genes
• spliced to produce continuous polypeptide coding
  sequence

65
Learning Objective 11

• What is the difference between translation in
  bacterial and eukaryotic cells?

66
Bacterial Cells

• Transcription and translation are coupled
• Bacterial ribosomes
• bind to 5' end of growing mRNA
• initiate translation before message is fully
  synthesized

67
Bacterial mRNA
68

Promoter region
mRNA termination sequence
Transcribed region
DNA
Upstream leader sequences
Downstream trailing sequences
Protein-coding sequences Translated region
Start codon
Stop codon
mRNA
OH 3 ' end
5 ' end
Polypeptide
Fig. 13-10, p. 288
69
Initiation
70

Leader sequence
mRNA
Initiation factor
Small ribosomal subunit
Start codon
Fig. 13-13a, p. 291
71

fMet
Initiator tRNA
Fig. 13-13b, p. 291
72

P site
Large ribosomal subunit
fMet
E site
A site
Initiation complex
Fig. 13-13c, p. 291
73
Elongation
74

tRNA with an amino acid
Amino acids
Amino acids
GDP
GTP
E
P
A
P
A
E
Aminoacyl-tRNA binds to codon in A site
mRNA
Ribosome ready to accept another aminoacyl-tRNA
Peptide bond formation
Amino end of polypeptide
New peptide bond
Translocation toward 3 ' end of mRNA
E
P
A
E
P
A
GTP
GDP
Fig. 13-14, p. 292
75
Termination
76

Release factor
E
P
A
mRNA
Stop codon (UAA, UAG, or UGA)
Fig. 13-15a, p. 293
77

Polypeptide chain is released
Stop codon (UAA, UAG, or UGA)
Fig. 13-15b, p. 293
78

Large ribosomal subunit
E
P
A
Release factor
mRNA
Small ribosomal subunit
tRNA
Fig. 13-15c, p. 293
79
Polyribosome

• Many ribosomes bound to a single mRNA

80
KEY CONCEPTS

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

81
Learning Objective 12

• Describe retroviruses and the enzyme reverse
  transcriptase

82
Retroviruses

• Synthesize DNA from an RNA template
• HIV-1 (virus that causes AIDS)
• Enzyme reverse transcriptase
• reverses flow of genetic information

83
Reverse Transcription
84

Chromosome DNA in nucleus of host cell
Provirus inserted into chromosome DNA
DNA provirus
DNA replication
Digestion of RNA strand
RNA /DNA hybrid
Reverse transcription
Viral RNA
RNA virus
Fig. 13-19a, p. 297
85

Provirus DNA transcribed
Viral mRNA
Viral RNA
Viral proteins
RNA virus
2
Fig. 13-19b, p. 297
86
Learning Objective 13

• Give examples of the different classes of
  mutations that affect the base sequence of DNA
• What effects does each have on the polypeptide
  produced?

87
Base Substitution

• May alter or destroy protein function
• missense mutation
• codon change specifies a different amino acid
• nonsense mutation
• codon becomes a stop codon
• May have minimal effects
• if amino acid is not altered
• if codon change specifies a similar amino acid

88
Normal DNA sequence
Normal mRNA sequence
Normal protein sequence
(Stop)
BASE-SUBSTITUTION MUTATIONS
Missense mutation
(Stop)
Nonsense mutation
(Stop)
Fig. 13-20a, p. 299
89
Animation Base-Pair Substitution
CLICKTO PLAY
90
Frameshift Mutations

• Insertion or deletion of one or two base pairs in
  a gene
• destroys protein function
• changes codon sequences downstream from the
  mutation

91
Normal DNA sequence
Normal mRNA sequence
(Stop)
Normal protein sequence
FRAMESHIFT MUTATIONS
Deletion causing nonsense
(Stop)
Deletion causing altered amino acid sequence
Fig. 13-20b, p. 299
92
Animation Frameshift Mutation
CLICKTO PLAY
93
Transposons

• Movable DNA sequences
• jump into the middle of a gene
• Retrotransposons
• replicate by forming RNA intermediate
• reverse transcriptase converts to original DNA
  sequence before jumping into gene

94
KEY CONCEPTS

• Mutations can cause changes in phenotype

95
Animation Protein Synthesis Summary
CLICKTO PLAY