Transcript Chapter 9 - Proteins and their synthesis

Chapter 9 Proteins and Their Synthesis
Green Fluorescent Protein drawn in cartoon style with fluorophore highlighted as ball-and-stick;
one wholly-reproduced protein, and cutaway version to show the fluorophore.
Review
Central Dogma
DNA
5’ ATG GAC CAG TCG GTT TAA GCT 3’
3’ TAC CTG GTC AGC CAA ATT CGT 5’
transcription
RNA
5’ AUG GAC CAG UCG GUU UAA GCU 3’
translation
Protein
aa - aa - aa - aa - aa - aa - aa
Protein Structure
via condensation
Protein Structure
Primary Structure
Protein folding is dependent on
the amino acid R groups
R
General Structure
H2N
C
COOH
H
There are 20 amino acids.
Their properties are determined by the R group.
There are 20 amino acids.
 Nonpolar or hydrophobic (9)
 Polar (hydrophillic), but uncharged (6)
 Polar (hydrophillic), but charged (5)
Nonpolar
ring
(Hydrophobic)
sulfur
Protein Structure
Primary Structure
Protein Structure
Two major types
of Secondary
Structure
α Helix
β Sheet
Protein Structure
How do we get from DNA to Primary
protein structure ?
DNA
5’ ATG GAC CAG TCG GTT TAA GCT 3’
3’ TAC CTG GTC AGC CAA ATT CGT 5’
transcription
RNA
5’ AUG GAC CAG UCG GUU UAA GCU 3’
translation
Protein
aa - aa - aa - aa - aa - aa - aa
DNA (mRNA) is read in Triplets
-Codon – Group of 3 DNA bases codes for a specific
amino acid
Ex. ATG = methionine
-This means the code is degenerate – more than one
codon can specify one amino acid
The Genetic Code - Nonoverlapping
Key To The Genetic Code
Groups of 3 mRNA
bases (codons) code for
specific amino acids
5’ CCAACCGGG 3’
CCA-ACC-GGG
Pro-Thr-Gly
The Genetic Code – Stop Codons
UGA
UAA
UAG
Proteins and Genes are Colinear
Mutations in DNA show specific corresponding changes
in the protein
Genes are converted to proteins in a linear fashion
Key To The Genetic Code
CCG UGG AGA GAC UAA
Pro – Trp – Arg –Asp - Stop
CCG UCG AGA GAC UAA
Pro – Ser – Arg –Asp - Stop
CCG UGG CGA GAC UAA
Pro – Trp – Arg –Asp - Stop
CCG UGG AGA GAC UAA
Pro – Stop
CCG UGG AGA CGA CUA
Pro – Trp – Arg –Arg - Leu
The Genetic Code - Mutations
4 Types of Mutations
1. Silent mutations
2. Missense mutations
3. Nonsense mutations
4. Frameshift mutations
The Genetic Code
mRNA has 3 potential “reading frames”
5’ CUUACAGUUUAUUGAUACGGAGAAGG 3’
3’ GAAUGUCAAAUAACUAUGCCUCUUCC 5’
5’ CUU ACA GUU UAU UGA UAC GGA GAA GG 3’
3’ GAA UGU CAA AUA ACU AUG CCU CUU CC 5’
5’ C UUA CAG UUU AUU GAU ACG GAG AAG G 3’
3’ G AAU GUC AAA UAA CUA UGC CUC UUC C 5’
5’ CU UAC AGU UUA UUG AUA CGG AGA AGG 3’
3’ GA AUG UCA AAU AAC UAU GCC UCU UCC 5’
Stop
UAA
UGA
UAG
The Genetic Code
mRNA has 3 potential reading frames
5’ CUUACAGUUUAUUGAUACGGAGAAGG 3’
3’ GAAUGUCAAAUAACUAUGCCUCUUCC 5’
5’ CUU ACA GUU UAU UGA UAC GGA GAA GG 3’
3’ GAA UGU CAA AUA ACU AUG CCU CUU CC 5’
5’ C UUA CAG UUU AUU GAU ACG GAG AAG G 3’
3’ G AAU GUC AAA UAA CUA UGC CUC UUC C 5’
5’ CU UAC AGU UUA UUG AUA CGG AGA AGG 3’
3’ GA AUG UCA AAU AAC UAU GCC UCU UCC 5’
Stop
UAA
UGA
UAG
Review - RNA
mRNA- messenger RNA
tRNA- transfer RNA
rRNA- Ribosomal RNA
tRNA-The adapter
tRNA-The adapter
•-tRNA functions as the adapter
between amino acids and the
RNA template
•-tRNAs are structurally similar
except in two regions
•Amino acid attachment site
•Anticodon
tRNA-The anticodon
The tRNA anticodon
•3 base sequence
•Complementary to the codon
•Base pairing between the mRNA and the tRNA
•Oriented and written in the 3’ to 5’ direction
tRNA
Aspartic Acid
3’ CUG 5’
5’ GAC 3’
mRNA
Aminoacyl-tRNA
synthetase
The enzyme responsible
for joining an amino
acid to its
corresponding tRNA
20 tRNA synthetases –
1 for each amino acid
Wobble
Allows one tRNA to recognize multiple codons
Occurs in the 3rd nucleotide
of a codon
Wobble – A new set pairing of rules
I = Inosine: A rare base found in tRNA
Wobble – A new set pairing of rules
Isoaccepting tRNAs: tRNAs that accept the same
amino acid but are transcribed from different genes
Wobble Problem
What anticodon would you predict for a tRNA species carrying
isoleucine?
Ribosomes – General characteristics
•Come together with tRNA and mRNA to create protein
•Ribosome consist of one small and one large subunit
•In prokaryotes, 30S and 50S subunits form a 70S
particle
•In Eukaryotes, 40S and 60S subunits form an 80S
particle
•Each subunit is composed of 1 to 3 types of rRNA and
up to 49 proteins
Ribosomes – General characteristics
Ribosomes – General characteristics
• rRNA folds up by
intramolecular base
pairing
Ribosomes – General characteristics
Translation
Synthesizing Protein
An overview
Translation Initiation - Prokaryotes
Translation begins at an AUG codon – Methionine
Requires a special “initiator” tRNA charged with
Met – tRNAMeti
This involves the addition of a formyl group to
methionine while it is attached to the
initiator
Shine-Dalgarno Sequence
mRNA only associates with unbound 30S subunit
Translation Initiation – Prokaryotes
Initiation Factors
3 initiation factor proteins are required for the start of
translation in prokaryotes
IF1 – Binds to 30S subunit as part of the complete initiation
complex. Could be involved in stability
IF2 – Binds to charged initiator tRNA and insures that other
tRNAS do not enter initiation complex
IF3 – Keeps the 30S subunit disassociated from the 50S subunit
and allows binding of mRNA
Figure 2-12-1
Figure 9-15-1
Figure 2-12-1
Figure 9-15-2
Figure 2-12-1
Figure 9-15-3
Translation Initiation – Eukaryotes
1. mRNA is produced in the nucleus and
transported to the cytoplasm
2. 5’ end of the mRNA is “capped” to prevent
degradation
3. Eukaryotic Initiation Factors (eIF4A, eIF4B,
and eIF4G) associate with the 5’ cap, the 40S
subunit, and initiator tRNA
4. Complex moves 5’ to 3’ unwinding the mRNA
until an initiation site (AUG) is discovered
5. Initiation factors are released and 60S
subunit binds
Figure 2-12-1
Figure 9-16-1
1. mRNA is produced in the
nucleus and transported to
the cytoplasm
2. mRNA is covered with
proteins and often folds on
itself
3. 5’ end of the mRNA is
“capped” to prevent
degradation
Figure 2-12-1
Figure 9-16-2
4.Eukaryotic Initiation
Factors (eIF4A,
eIF4B, and eIF4G)
associate with the 5’
cap, the 40S subunit,
and initiator tRNA
Figure 9-16-3
5. Complex moves 5’ to
3’ unwinding the
mRNA until an
initiation site (AUG)
is discovered
Figure 9-16-4
6. Initiation factors
are released and 60S
subunit binds
Elongation
• Requires two protein Elongation Factors:
EF-Tu and EF-G
•
Amino acids are added to the growing peptide chain
at the rate of 2-15 amino acids per second
Elongation
Termination
Release Factors – RF1, RF2 and RF3
•RF1 recognizes UAA or UAG
•RF2 recognizes UAA or UGA
•RF3 assists both RF1 and RF2
Stop codon also called a nonsense codon
A water molecule in the
peptidyltransferase center leads to the
release of the peptide chain
Translation differences between
Eukaryotes and Prokaryotes
Prokaryotes
Eukaryotes
• NO nuclear membrane
• Translation coupled to
transcription
• Presence of a nuclear membrane
• Ribosomes bind the Shine
Dalgarno sequence
• mRNA can contain multiple
genes
• Ribosome binds to the 5’ cap
• mRNA has information for
only one gene
• Formylmethionine bound to
initiator tRNA
• mRNA exported from
nucleus
• Methionine bound to initiator
tRNA
Posttranslational Folding
Proteins must fold correctly to be functional
Correct folding is not always energetically favorable in the
cytoplasm
Chaperones (including GroE chaperonins) bind to nascent
peptides and facilitate correct folding
Posttranslational modifications
Phosphorylation
Many proteins require some
type of modification to become
functional
Posttranslational modifications
Glycosylation – adding sugars
Signaling molecules
Cell wall proteins
Glycoproteins
Posttranslational modifications
Ubiquitination marks a protein
for degradation
-Short lived proteins
(functional in cell cycle)
- Damaged or mutated proteins
Summary
• Translation
– Prokaryote
– Eukaryote
• Post translational modifications
– Phosphorylation
– Glycosylation
– Ubiquitination