dna replication & protein synthesis
DESCRIPTION
DNA Replication & Protein Synthesis. Structure of DNA & RNA. DNA and RNA. Deoxyribonucleic acid - DNA Ribonucleic acid - RNA Both made of nucleotides Nucleotide building blocks: sugar + phosphate + base. Sugars. 5 carbon sugar DNA’s sugar is deoxyribose - PowerPoint PPT PresentationTRANSCRIPT
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DNA Replication & Protein Synthesis
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Structure of DNA & RNA
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DNA and RNA
Deoxyribonucleic acid - DNA
Ribonucleic acid - RNA
Both made of nucleotides
Nucleotide building blocks:
sugar + phosphate + base
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Sugars
5 carbon sugar
DNA’s sugar is deoxyribose
RNA’s sugar is ribose
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Two Classes of Bases
Purines: 2 rings
adenine
guanine
Pyrimidines: 1 ring
cytosine
thymine
Base always attaches to the #1 carbon on the sugar
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Phosphate
Always attaches to the #5 carbon on the sugar
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Watson & Crick Model for DNA
Two strands of nucleotides that form a double helix fig. 16.72 strands join in an antiparallel arrangementSugar & phosphate make the backbone while bases are held together by H-bondsBase pairs are always formed betweenA - TC - G
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DNA Replication
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DNA Replication
Each strand acts as a template for a new strand
Complimentary base pairing forms new strand
Called semi-conservative replication -- Why?
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Meselson-Stahl Experiment
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Replication in E.coli
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Replication in Eukaryotes
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Comparison
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Enzymes involved
Single strand binding protein - holds site open
Helicase – breaks helix
Topoisomerase – prevents supercoiling
Primase – initiates the RNA primer
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Enzymes cont’d
DNA polymerase cannot initiate synthesis.
An RNA primer is needed.
RNA primer is later replaced by DNA.
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Replication in eukaryotes
1. H-bonds break at origin of replication
2. Replication bubble forms as H-bonds break
3. DNA polymerase directs synthesis of new strands
4. Replication is bi-directional (proceeds in both directions) fig. 16.17
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Replication cont’d
5. DNA polymerase can only build the new strand in the in 5' 3' direction therefore new nucleotides are only added to the existing 3' sideOne strand is synthesized continuously - leading strandOne strand synthesized in pieces -- lagging strand pieces called Okazaki fragments
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Replication cont’d
6. Okazaki fragments joined by DNA ligase
7. DNA polymerase proofreads
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Replication Animations
http://www.fed.cuhk.edu.hk/~johnson/teaching/genetics/animations/dna_replication.htm
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html
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Replication cont’d
8. Energy required to build new strand
provided by ATP-like molecules:
3 PO4’s, 1 deoxyribose, 1 base
DATP
DGTP
DTTP
DCTP
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Chromosome 11
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Gene Expression
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Gene Expression
AKA protein synthesis
Background:
- genes on chromosomes contain DNA
- each gene codes for one protein
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Two Stages of Protein Synthesis
1. Transcription
2. Translation
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Transcription
Production of mRNA (messenger RNA) from DNA
RNA similar to DNA except:
- ribose instead of deoxyribose
- uracil instead of thymine
- single stranded
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Steps of Transcription
1. Initiation
2. Elongation
3. Termination
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Steps of Transcription cont’d
1. Helicase breaks H-bonds
2. One strand of DNA serves as template for mRNA
3. Uses RNA polymerase
4. Synthesis in 5' 3' direction
5. mRNA leaves nucleus
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RNA Processing
Occurs in the nucleus
Addition of 5’ cap and poly-A-tail
Splicing
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Cap and tail
Aids in export from nucleus
Protects RNA from degradation
\once in cytoplasm these along with cytoplasmic proteins help ribosome attachment.
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RNA Splicing
Why ?
Some sequences of DNA don’t code for anything & are b/w ones that do.
Noncoding segment called introns
Coding segment called exons
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What Happens?
mRNA made in nucleus is pre mRNARNA splicing takes out introns & puts exons as a continuous strand snRNP’s (snurps) proteins & RNA at end of proteins snRNP’s & other proteins form a spliceosome -- where splicing occurs Pg. 312 fig. 17.10
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Translation
Interpreting amino acid sequence from nucleotide language
Proteins made according to codons
Codons - 3 nucleotide sequence on mRNA
Each codon specifies one amino acid
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Codons read in 5' 3' direction
AUG is start codon
Use chart pg. 308 to determine the amino acid coded for by each codon -- (mRNA)
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2 other RNA’s needed
tRNA
rRNA
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tRNA
Carries amino acid to ribosome – see structure fig. 17.13
A.a. attached to 3' end
Anticodon read 3' 5'
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rRNA
Component of ribosome – maintains structure of ribosome as well as regulation of mRNA & tRNA
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Ribosome Structure
Two subunits -- small & large
Lg. Unit has three sites
- A site (aminoacyl)
- P site (peptidyl)
- E site (exit)
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3 Phases of Translation
1. Initiation
2. Elongation
3. Termination
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Initiation
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Initiation
1. 5' end of mRNA attaches to small subunit of ribosome
2. Start codon, AUG, binds w/ initiator tRNA (met)
3. P – site of lg. subunit binds to AUG mRNA codon
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Elongation
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Elongation
1. 2nd tRNA enters A- site & binds to 2nd codon
2. Peptide bond forms b/w a.a. of each tRNA3. 1st tRNA moves from P-site to E-site4. As mRNA moves through ribosome 2nd
tRNA now in A-site w/ 2 a.a.’s5. Cycle repeats until a STOP codon enters
A-site
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Termination
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Termination
1. STOP codon in A-site
2. Protein release factor binds to codon -- no tRNA -- no a.a.
3. Polypeptide is freed
4. Two subunits separate
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Trivial but Important
Some tRNA’s have anticodons that can recognize 2 or more different codons
Third base of codon & anticodon can vary
I.e. U can bind w/ either A or G
This is called wobble
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Folding and Modification
Some amino acids can be modified by attaching sugars, lipids phosphate groups etc.
Enzymes may remove some amino acids from leading end
All translation starts with a free ribosome and then depending on the developing polypeptide chain it may attach to rough ER
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Folding and Modification
Polypeptides of proteins destined for endomembrane system (secretion) are marked by a signal peptide (directs it to rough ER)
Signal peptide is recognized by a protein-RNA complex called a signal recognition particle (SRP)
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Regulation of Gene Expression
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Regulation in Prokaryotes
Operon Theory
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Review transcription
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Operon Structure
Promoter – where transcription begins
TATA box
Operator – on/off switch
Structural genes – code for polypeptide
Terminator – stop sequence
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Two types of operons
1. Synthesis of repressible enzymes
2. Synthesis of inducible enzymes
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Repressible
Tryp operon fig. 18.20Alone the operator is on & tryptophan is producedAs tryptophan accumulates it binds to the repressorRepressor now fits into operator and blocks attachment of RNA polymerase – operator is now off
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Inducible
Lac operon fig. 18.21
When no lactose present active repressor fits into operator thus keeping it off
Lactose present & changes to allolactose, an isomer
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Allolactose binds to repressor and inactivates it
Enzymes for lactose breakdown are produced
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Regulation in eukaryotes
Histone modification
Methylation of DNA
Chromatin structure
Initiation of transcription
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Histone
Small protein with a high proportion of positive charged amino acids that bind to negative DNA
Role is chromatin structure
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Mutations
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Mutations
Any change in sequence of DNA
Most mutations are harmless b/c only 10-20% of all human DNA actually codes for proteins -- some junk DNA present
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2 Types of Mutations
Large -- delete or rearrange pieces or whole chromosomes
Small -- single nucleotide change called point mutation
- SNPs single-nucleotide polymorphism
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SNPs
http://www.ncbi.nlm.nih.gov/About/primer/snps.html
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2 Types of Point Mutations
Substitution
-- Only one amino acid is affected
-- I.e. Sickle celled anemia
-- Missense change one amino acid to another
-- Sometimes has no effect on amino acid
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Fig. 17-22
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
33
3
3
3
3
55
5
55
5
C CT T TTG GA A AA
A A AGG U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
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Fig. 17-23Wild-type
3DNA template strand
5
5
53
3
Stop
Carboxyl endAmino end
Protein
mRNA
33
3
55
5
A instead of G
U instead of C
Silent (no effect on amino acid sequence)
Stop
T instead of C
33
3
55
5
A instead of G
Stop
Missense
A instead of T
U instead of A
33
3
5
5
5
Stop
Nonsense No frameshift, but one amino acid missing (3 base-pair deletion)
Frameshift causing extensive missense (1 base-pair deletion)
Frameshift causing immediate nonsense (1 base-pair insertion)
5
5
533
3
Stop
missing
missing
3
3
3
5
55
missing
missing
Stop
5
5533
3
Extra U
Extra A
(a) Base-pair substitution (b) Base-pair insertion or deletion
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Fig. 17-23aWild type
3DNA templatestrand
3
355
5mRNA
Protein
Amino end
Stop
Carboxyl end
A instead of G
33
3
U instead of C
55
5
Stop
Silent (no effect on amino acid sequence)
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Fig. 17-23bWild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
T instead of C
A instead of G
33
3
5
5
5
Stop
Missense
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Fig. 17-23cWild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
A instead of T
U instead of A
33
3
5
5
5
Stop
Nonsense
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Fig. 17-23dWild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
Extra A
Extra U
33
3
5
5
5
Stop
Frameshift causing immediate nonsense (1 base-pair insertion)
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Fig. 17-23eWild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
missing
missing
33
3
5
5
5
Frameshift causing extensive missense (1 base-pair deletion)
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Fig. 17-23fWild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
missing
missing
33
3
5
5
5
No frameshift, but one amino acid missing (3 base-pair deletion)
Stop
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Addition or deletion
-- Also called frame shift mutation. Why?
-- Changes all codons after mutation
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Example
Normal sequence
THE FAT CAT ATE ONE ANT AND ONE NUT
Substitution
THE FAT CAN ATE ONE ANT AND ONE NUT
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More ExamplesDeletion
THE FAT CA_A TEO NEA NTA NDO NEN UT…Addition
THE FAT CAT ART EON EAN TAN DON ENU T…Addition and Deletion
THE FAT CA_A RTE ONE ANT AND ONE NUT
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Genetic Engineering
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Genetic Engineering
Terms
Plasmid
– extra circular DNA in some bacteria
Restriction Enzymes
– Enzymes found in bacteria that cut up foreign DNA Why?
protection
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How?
Recognizes a specific sequence of 4-8 nucleotides
Cuts DNA at that sequence
Bacteria protects itself from restriction by adding CH3 groups to adenine or cytosine
This keeps restriction enz. from recognizing itself
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Restriction Enzymes Are Useful
Sticky ends are produced when DNA is cut.These ends can now join to new DNA of choice
DNA ligase makes it permanentDNA can then be sent by a vector to enter new cellNew cell is then clonedSee fig. 20.1 and 20.3
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plasmids
http://www.dnalc.org/resources/plasmids.html
electrophoresis
http://learn.genetics.utah.edu/content/labs/gel/
You tube electrophoresis
http://www.youtube.com/watch?v=qMxQ-65qYDk
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Lab #6 Part A
Bacterial transformation with ampicillin resistance
Inserting a plasmid w/gene for ampicillin resistance into E. coli
-- pAMP is the plasmid w/ampicillin resistance
-- Luria broth is food for the bacteria
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We will try to put the plasmid into the E. coli
How will we know if it worked?
Grow E. coli on ampicillin agar plates & measure growth
We then calculate the efficiency rate.
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Lab #6 Part B
Electrophoresis – tool for use with DNA
-- operates with a gel and electricity
-- separates fragments of DNA by size
-- can be used to identify individuals
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Lab 6BWe will use electrophoresis to find if the suspect of a crime is the actual criminalLab 6 has us use electrophoresis to find the number of base pairs in each fragment of DNA
-- this is done by sending known DNA fragments alongside of unknown DNA fragments
-- then measure the distance each fragment traveled
-- use interpolation technique on a graph to find the actual number of base pairs in each fragment
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Polymerase Chain Reaction
Method used to make many copies of a single strand of DNA
Uses a DNA polymerase that can withstand the heat used to separate DNA
Very useful when DNA is in short supply
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