Making Proteins
TRANSLATION
TRANSCRIPTION
Protein
mRNACytoplasm
mRNA
DNA
Nucleus Informationstorage
Informationcarrier
Active cellmachinery
Central Dogma of Genetics
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).
There are 4 RNA bases (U, C, A, G) and they must specify 20 amino acids.
How many bases specifya single amino acid?
Since there are only 4 bases, a singlet codecould onlyspecify 4amino acids.
A doublet code couldspecify a maximum of4 x 4 or 16 amino acids.
A triplet code could specify amaximum of 4 x 4 x 4, or 64amino acids.
1 Base? 2 Bases? 3 Bases? 4 Bases?...
4 < 20: Not enough
16 < 20: Not enough 64 > 20: More than enough
1 2 3 4 1 2 3 4
5 6 7 8
9 10 11 12
13 14 15 16
1 2 3 4
5 6 7 8
9 10 11 12
13 14 15 etc...
U GC A U
G
C
A
U
G
C
A
U
G
C
A
U
G
C
A
U
G
C
A
U
G
C
A
U
G
C
A
U
C
A
U C A G
U C A G
U C A G
U C A G
U C A G
U C A G
U C A G
U C A
U
G
C
A
U
G
C
A
U
G
C
A
U
C
A
mRNAGC CA
CC
G AGA A AA AA A AA AU U U UU U U U UC CC CC CC CCC C CGG G
GG G
G
G
How does the genetic code work?
Figure 17.4 The dictionary of the genetic code
One gene-one polypeptide hypothesis:
A gene is a length of a DNA molecule that
contains the information to produce one polypeptide
chain
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 1)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 2)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 3)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 4)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 5)
5’
3’5’
3’
3’
5’
3’5’
DNARNA
PP
P
O
OH
OH
O
OH
OH 3’P P PP
5’
RN
A
OHO
OOH
OH
AU
A T CG
C
5’3’
O O O O
P P P PG
DN
A
Transcription produces an RNA molecule complementary to a DNA template
Template strand
RNApolymerase DNA
RNA transcription is catalyzed by RNA polymerase
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'.
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 1)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 2)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 3)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 4)
Close up of transcription
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
In eukaryotes: proteins called transcription factors bind to the promoter first, then RNA polymerase binds to start transcription
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--> 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.
RNA processing in Eukaryotes
Molecules called small nuclear ribonucleoproteins (snRNPs) combine to splice
introns from mRNA
Figure 17.11 Correspondence between exons and protein domains
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
Active site(containsonly rRNA)
A site
P site
Large subunitSmall subunit
rRNAs = ribosomal RNA
Proteins
Structure of a ribosome
E site
Loops consistof unpairedbases
The adaptor molecule between mRNA and protein is tRNA (transfer RNA)
Stems are createdby hydrogen bondingbetween complementarybase pairs
Figure 17.13b The structure of transfer RNA (tRNA)
An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA
Ser
ACC
3’
5’Binding site foramino acid
Binding site formRNA codon
Aminoacid
GA U
U CA
Serine anticodon
Serine codon
Early model of tRNA function
5’ 3’
mRNA
Figure 17.15 The anatomy of a functioning ribosome
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.
Figure 17.17 The initiation of translation
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.
Figure 17.18 The elongation cycle of translation
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.
Figure 17.19 The termination of translation
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
Virus protein coat
Host cell membrane
Virus RNA
1. Start of infection.Virus RNA enters hostcells.
2. Reverse transcriptase uses Virus RNA as template to produce virus DNA
4. End of infection.New generation ofvirus particles burstfrom host cell.
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.
3. Virus DNA directsthe production of newvirus particles.
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.
A A C T G G C
T T G A C C G
A A C T G G C
T T G A T C G
T T G A C C G
A A C T G G C
A A C T G G C
T T G A C C G
T T G A T C G
A A C T A G C
A A C T G G C
T T G A C C G
T T G A C C G
A A C T G G C
Parental DNA
DNA replication
First generation progeny
3'
5'
5'
3'
Second generation progeny
Wild type
MUTANT
Wild type
Wild type
DNA replication
Point mutations are a change in single base pair of DNA
A base-pair substitution:
Figure 17.24 Categories of Base-pair substitutions
DNA sequence
Amino acid sequence
DNA sequence
Amino acid sequence
CACGTG
GTGCAC
GACCTG
TGAACT
GGACCT
CTCGAG
CTCGAG
CACGTG
GTGCAC
GACCTG
TGAACT
GGACCT
CTCGAG
ValineHistidine
LeucineThreonine
Proline Glutamic acid
Glutamic acid
CACGTG
ValineHistidine
LeucineThreonine
Proline ValineGlutamic acid
Normal
Mutant
Start of coding sequence
Normal red blood cells
Sickled red blood cells
DNA point mutations can lead to a different amino acid sequence.
Phenotype
Insertion or deletion of a single base-pair causes
frameshift mutations
DNA strand with adjacent thymine bases
P
CH2
P
CH2
P
O
O N
N
O
H CH3
O
HN
O
H CH3
O
HN
Thymine
Thymine
UV light
P
CH2
P
CH2
P
O
ON
N
O
H CH3
O
HN
O
H CH3
O
HN
Thymine dimer
Kink
UV-induced thymine dimers caused DNA to kink
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
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.
3'
5'
3'
5'
T G T C CA
T C G C
A C A G GG
OH 3'
T G T C C A T C G C
A C A G G
GT
5'
5'
OH 3'
OH
Mismatched bases.
Polymerase III can repair mismatches.
DNA polymerase proofreads DNA during replication
1. Where a mismatch occurs, the correct base is located on the methylated strand: the incorrect base occurs on the unmethylated strand.
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.
METHYLATION-DIRECTED MISMATCHED BASE REPAIR
Mismatch
Repaired Mismatch
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