Making Proteins

1

• Making Proteins

2
Central Dogma of Genetics
3
The Genetic Code

• The nucleotide sequence of DNA is a code DNA is
  an information-storage molecule without enzymatic
  capabilities(F. Crick).
• The information in DNA is copied into RNA, which
  is used to make proteins (mRNA messenger RNA).
• Hypothesis each of the 20 amino acids in
  proteins is specified by one or more 3 base
  codons (Gamow).

4
How does the genetic code work?
There are 4 RNA bases (U, C, A, G) and they must
specify 20 amino acids.
How many bases specify a single amino acid?
G
G
A
U
C
C
G
A
A
U
G
C
C
G
G
U
C
A
A
U
mRNA
G
A
A
U
U
C
C
G
A
A
A
A
U
U
G
A
C
C
C
C
C
G
G
C
U
C
C
C
3 Bases?
1 Base?
2 Bases?
4 Bases?...
A doublet code could specify a maximum of 4 x 4
or 16 amino acids.
A triplet code could specify a maximum of 4 x 4 x
4, or 64 amino acids.
U
U
U
U
U
C
A
G
U
U
U
U
U
U
U
U
U
C
A
G
G
C
A
U
2
2
3
4
1
1
3
4
1
2
3
4
C
C
C
C
C
C
C
C
U
C
A
G
U
C
A
G
C
C
C
C
4 lt 20 Not enough
5
6
7
8
6
5
7
8
A
A
A
A
A
U
C
A
G
A
A
A
A
A
U
C
A
G
A
A
Since there are only 4 bases, a singlet
code could onlyspecify 4 amino acids.
9
9
10
11
12
10
11
12
G
G
C
A
G
G
G
U
G
G
G
U
C
A
G
G
G
etc...
14
14
13
15
16
13
15
16 lt 20 Not enough
64 gt 20 More than enough
5
Figure 17.4 The dictionary of the genetic code
6
One gene-one polypeptide hypothesisA gene is a
length of a DNA molecule that contains the
information to produce one polypeptide chain
7
Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 1)
8
Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 2)
9
Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 3)
10
Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 4)
11
Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 5)
12
Transcription produces an RNA molecule
complementary to a DNA template
DNA
5
RNA
3
3
5
Template strand
5
3
3
5
P
OH
P
OH
P
O
G
OH
P
P
P
5
OH
3
P
RNA
OH
O
O
O
HO
OH
A
U
C
A
T
C
G
DNA
O
O
O
O
P
P
P
P
5
3
13
RNA transcription is catalyzed by RNA polymerase
RNA polymerase
DNA
14
Protein Synthesis Begins with the Process ofGene
Transcription

• Steps of Transcription
• RNA polymerase binds to the promoter region of
  the DNA
• RNA polymerase unwinds the DNA.
• RNA polymerase reads DNA 3' to 5' and synthesizes
  complementary RNA 5' to 3'.

15
Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 1)
16
Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 2)
17
Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 3)
18
Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 4)
19
Close up of transcription
20
In eukaryotes proteins called transcription
factors bind to the promoter first, then RNA
polymerase binds to start transcription
21
After Transcription

• Transcription in Prokaryotes
• The RNA produced is ready to be translated mRNA
• Transcription in Eukaryotes
• The RNA produced must be modified before
  translation 1 transcript--gt mRNA
• Eukaryotic mRNAs are processed in the nucleus by
  additionof a 5' cap and 3' poly A tail
• Eukaryotic genes have introns non-coding regions
  thatmust be removed from the primary mRNA to
  make an intact uninterrupted message.

22
RNA processing in Eukaryotes
23
Molecules called small nuclear ribonucleoproteins
(snRNPs) combine to splice introns from mRNA
24
Figure 17.11 Correspondence between exons and
protein domains
25
After transcription, the next step is translation

• Translation Converts the Nucleotide Sequence of
  mRNA into the Amino Acid Sequence of a Protein
• Translation occurs on ribosomes either in the
  cytoplasm or on the endoplasmic reticulum

26
Structure of a ribosome
Large subunit
Small subunit
P site
E site
Proteins
A site
Active site (contains only rRNA)
rRNAs ribosomal RNA
27
The adaptor molecule between mRNA and protein is
tRNA (transfer RNA)
Stems are created by hydrogen bonding between
complementary base pairs
Loops consist of unpaired bases
28
Figure 17.13b The structure of transfer RNA
(tRNA)
29
An aminoacyl-tRNA synthetase joins a specific
amino acid to a tRNA
30
Early model of tRNA function
Amino acid
Ser
3
A
C
Binding site for amino acid
C
5
Binding site for mRNA codon
Serine anticodon
A
U
G
5
3
U
C
A
mRNA
Serine codon
31
Figure 17.15 The anatomy of a functioning
ribosome
32
Translation Converts the Nucleotide Sequence of
mRNA into the Amino Acid Sequence of a Protein

• Translation occurs in three steps
• Initiation the ribosome 30S subunit binds mRNA
  and movesto the AUG codon, which is the
  translation start site.
• The initiator methionine tRNA binds to the AUG
  start codon.
• The ribosome 50S subunit assembles so that the
  initiator tRNA and the AUG codon are in the P
  site.

33
Figure 17.17 The initiation of translation
34
Translation Converts the Nucleotide Sequence of
mRNA into the Amino Acid Sequence of a Protein

• Translation occurs in three steps
• Elongation amino acids are joined together and
  the ribosome moves to the next codon.
• New tRNAs enters the A site of the ribosome
• A peptide bond forms between the polypeptide on
  the tRNA inthe P site and the amino acid in the
  A site, which transfers the polypeptide to the A
  site tRNA.
• The ribosome moves along the mRNA in the 5' to 3'
  direction.

35
Figure 17.18 The elongation cycle of translation
36
Translation Converts the Nucleotide Sequence of
mRNA into the Amino Acid Sequence of a Protein

• Translation occurs in three steps
• Termination when a stop codon on mRNA is
  encountered in the A site, the completed
  polypeptide is released, and the ribosome
  disengages.
• Release factors are required.

37
Figure 17.19 The termination of translation
38
Post-translational events affect the structure,
activity, and destination of the protein

• Proteins must fold into their proper 3D
  structure.

Primary structure
Secondary structure
Tertiary structure
Quaternary structure
39
The Central Dogma Information Flows from DNA to
RNA to Proteins (F.Crick)

• Viruses that have RNA genomes contradict the
  centraldogma, but all cells conform to it.

Virus protein coat
Host cell membrane
Virus RNA
1. Start of infection. Virus RNA enters
host cells.
2. Reverse transcriptase uses Virus RNA as
template to produce virus DNA
4. End of infection. New generation of virus
particles burst from host cell.
3. Virus DNA directs the production of new virus
particles.
40
Mutation and DNA Repair Mechanisms

• Mutations are created by chemicals, radiation,
  errors in meiosis and mistakes in DNA
  replication.
• Mutations can be deleterious, beneficial, or
  silent.
• Mutations in an individual are usually
  deleterious, may cause disease and death.
• Mutations in a population are a source of genetic
  diversity that allows evolution to occur.

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Point mutations are a change in single base pair
of DNA
A
A
C
T
G
G
C
A base-pair substitution
Wild type
T
T
G
A
C
C
G
A
A
C
T
G
G
C
A
A
C
T
A
G
C
MUTANT
3'
5'
T
T
G
A
T
C
G
T
T
G
A
T
C
G
A
A
C
T
G
G
C
DNA replication
DNA replication
T
T
G
A
C
C
G
A
A
C
T
G
G
A
A
C
T
G
G
C
C
5'
3'
Wild type
Parental DNA
T
T
G
A
C
C
G
T
T
G
A
C
C
G
First generation progeny
A
A
C
T
G
G
C
Wild type
T
T
G
A
C
C
G
Second generation progeny
42
Figure 17.24 Categories of Base-pair
substitutions
43
DNA point mutations can lead to a different amino
acid sequence.
Phenotype
Start of coding sequence
CAC
GTG
GAC
TGA
GGA
CTC
CTC
DNA sequence
GTG
CAC
CTG
ACT
CCT
GAG
GAG
Normal
Amino acid sequence
Normal red blood cells
Histidine
Threonine
Glutamic acid
Glutamic acid
Valine
Leucine
Proline
CAC
GTG
GAC
TGA
GGA
CTC
CAC
DNA sequence
GTG
CAC
CTG
ACT
CCT
GAG
GTG
Mutant
Amino acid sequence
Sickled red blood cells
Threonine
Histidine
Glutamic acid
Leucine
Valine
Valine
Proline
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Insertion or deletion of a single base-pair
causes frameshift mutations
45
UV radiation can cause 2 thymines that are next
to each other to bind to each other instead of
the adenines in the other strand
UV-induced thymine dimers caused DNA to kink
H
P
O
Thymine dimer
P
H
N
O
CH2
CH2
O
N
N
O
Thymine
O
N
O
DNA strand with adjacent thymine bases
UV light
H
CH3
H
CH3
P
H
P
O
O
H
Kink
N
N
O
CH2
CH2
O
N
O
O
Thymine
N
H
CH3
H
CH3
P
P
46
Mutation and DNA Repair Mechanisms

• DNA Repair Mechanisms
• DNA polymerase proofreads and corrects point
  mutations during replication.
• Other excision repair systems scan newly formed
  DNA and correct remaining mutations.
• Repair enzymes identify the correct template
  strand by its methyl groups.
• Defects in repair system enzymes are implicated
  in a variety of cancers.

47
DNA polymerase proofreads DNA during replication
3'
5'
A
T
G
T
C
C
T
C
G
C
Mismatched bases.
A
C
A
G
G
G
5'
OH 3'
5'
3'
Polymerase III can repair mismatches.
T
G
T
C
C
A
T
C
G
C
A
C
A
G
G
5'
OH 3'
T
G
OH
48
METHYLATION-DIRECTED MISMATCHED BASE REPAIR
1. Where a mismatch occurs, the correct base is
located on the methylated strand the incorrect
base occurs on the unmethylated strand.
Mismatch
2. Enzymes detect mismatch and nick unmethylated
strand.
3. DNA polymerase I excises nucleotides on
unmethylated strand.
4. DNA polymerase I fills in gap in 5'    3'
direction.
5. DNA ligase links new and old nucleotides.
Repaired Mismatch
49
Some genetic diseases are associated with
mutations in DNA repair mechanisms
Xeroderma pigmentosum is a defect in ultraviolet
radiation induced DNA repair mechanisms
characterized by severe sensitivity to all
sources of UV radiation (especially sunlight).
Symptoms include blistering or freckling,
premature aging of skin,with increased cancers in
these same areas, blindness resulting from eye
lesions or surgery for skin lesions close to the
eyes