chapter 9 proteins and their synthesis
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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 PresentationTRANSCRIPT
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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.
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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
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Protein Structure
via condensation
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Protein Structure
Primary Structure
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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
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There are 20 amino acids.
Nonpolar or hydrophobic (9)
Polar (hydrophillic), but uncharged (6)
Polar (hydrophillic), but charged (5)
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Nonpolar
(Hydrophobic)ring
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sulfur
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Protein Structure
Primary Structure
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Protein Structure
Two major types of Secondary Structure
α Helix
β Sheet
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Protein Structure
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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
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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
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The Genetic Code - Nonoverlapping
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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
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The Genetic Code – Stop Codons
UGA
UAA
UAG
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Proteins and Genes are ColinearMutations in DNA show specific corresponding changes in the protein
Genes are converted to proteins in a linear fashion
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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
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The Genetic Code - Mutations
4 Types of Mutations
1. Silent mutations
2. Missense mutations
3. Nonsense mutations
4. Frameshift mutations
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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
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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
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Review - RNA
mRNA- messenger RNA
tRNA- transfer RNA
rRNA- Ribosomal RNA
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tRNA-The adapter
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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
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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
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Aminoacyl-tRNA synthetaseThe enzyme responsible for joining an amino acid to its corresponding tRNA
20 tRNA synthetases – 1 for each amino acid
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WobbleAllows one tRNA to recognize multiple codons
Occurs in the 3rd nucleotide of a codon
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Wobble – A new set pairing of rules
I = Inosine: A rare base found in tRNA
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Wobble – A new set pairing of rules
Isoaccepting tRNAs: tRNAs that accept the same amino acid but are transcribed from different genes
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Wobble ProblemWhat anticodon would you predict for a tRNA species carrying isoleucine?
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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
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Ribosomes – General characteristics
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Ribosomes – General characteristics
• rRNA folds up by intramolecular base pairing
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Ribosomes – General characteristics
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Translation
Synthesizing Protein
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An overview
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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
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Shine-Dalgarno Sequence
mRNA only associates with unbound 30S subunit
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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
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Figure 9-15-1
Figure 2-12-1
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Figure 9-15-2
Figure 2-12-1
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Figure 9-15-3
Figure 2-12-1
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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
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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
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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
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Figure 9-16-3
5. Complex moves 5’ to 3’ unwinding the mRNA until an initiation site (AUG) is discovered
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Figure 9-16-4
6. Initiation factors are released and 60S subunit binds
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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
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Elongation
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TerminationRelease 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
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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
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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
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Posttranslational modifications
PhosphorylationMany proteins require some type of modification to become functional
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Posttranslational modifications
Glycosylation – adding sugars
Signaling molecules
Cell wall proteins
Glycoproteins
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Posttranslational modifications
Ubiquitination marks a protein for degradation
-Short lived proteins (functional in cell cycle)
- Damaged or mutated proteins
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Summary• Translation
– Prokaryote– Eukaryote
• Post translational modifications– Phosphorylation– Glycosylation– Ubiquitination