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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. 5 ’ ATG GAC CAG TCG GTT TAA GCT 3 ’ - PowerPoint PPT Presentation
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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.
ReviewCentral Dogma
5’ ATG GAC CAG TCG GTT TAA GCT 3’3’ TAC CTG GTC AGC CAA ATT CGT 5’
aa - aa - aa - aa - aa - aa - aa
DNA
RNA
Protein
5’ AUG GAC CAG UCG GUU UAA GCU 3’
transcription
translation
Protein Structure
via condensation
Protein Structure
Primary Structure
Protein folding is dependent on the amino acid R groups
General Structure
There are 20 amino acids.
Their properties are determined by the R group.
R
H2N C COOH H
There are 20 amino acids.
Nonpolar or hydrophobic (9)
Polar (hydrophillic), but uncharged (6)
Polar (hydrophillic), but charged (5)
Nonpolar
(Hydrophobic)ring
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 ?
5’ ATG GAC CAG TCG GTT TAA GCT 3’3’ TAC CTG GTC AGC CAA ATT CGT 5’
aa - aa - aa - aa - aa - aa - aa
DNA
RNA
Protein
5’ AUG GAC CAG UCG GUU UAA GCU 3’
transcription
translation
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 UGG AGA GAC UAA
Pro – Stop
CCG UGG CGA GAC UAA
CCG UGG AGA CGA CUAPro – Trp – Arg –Arg - Leu
CCG UCG AGA GAC UAA Pro – Ser – Arg –Asp - Stop
Pro – Trp – Arg –Asp - Stop
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’
StopUAAUGAUAG
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’
StopUAAUGAUAG
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
3’ CUG 5’5’ GAC 3’
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
mRNAAspartic Acid
Aminoacyl-tRNA synthetaseThe 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 – tRNAMet
i
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 – ProkaryotesInitiation 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 9-15-1
Figure 2-12-1
Figure 9-15-2
Figure 2-12-1
Figure 9-15-3
Figure 2-12-1
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 9-16-1
Figure 2-12-1
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 9-16-2
Figure 2-12-1
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
A water molecule in the peptidyltransferase center leads to the release of the peptide chain
Stop codon also called a nonsense codon
Translation differences between Eukaryotes and Prokaryotes
• NO nuclear membrane• Translation coupled to transcription
• Ribosomes bind the Shine Dalgarno sequence• mRNA can contain multiple genes
• Formylmethionine bound to initiator tRNA
• Presence of a nuclear membrane
• mRNA exported from nucleus
• Ribosome binds to the 5’ cap
• mRNA has information for only one gene
• Methionine bound to initiator tRNA
Prokaryotes Eukaryotes
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