Post on 27-Jun-2015
description
DNA Replication and Protein Synthesis
Patricia Caldani MS
ObjectiveObjective
Genome structureGenome structure ChromosomeChromosome/Gene/DNA/Gene/DNA
DNA ReplicationDNA Replication Protein SynthesisProtein Synthesis
Transcription and TranslationTranscription and Translation MutationsMutations
The Human GenomeThe Human Genome The human genome is made up of The human genome is made up of
3 x 103 x 109 9 base pairs of DNA (haploid base pairs of DNA (haploid
genome)genome)
This contains 30,000 genes This contains 30,000 genes arranged on 46 chromosomesarranged on 46 chromosomes
Packaged within the nucleus of the Packaged within the nucleus of the cellcell
What is a Gene??What is a Gene?? Genes are instruction manuals for Genes are instruction manuals for
our bodiesour bodies They are the directions for building They are the directions for building
all the proteins that make our body all the proteins that make our body functionfunction
Genes are made up of DNAGenes are made up of DNA EXP. RBC use Hemoglobin EXP. RBC use Hemoglobin
ChromosomesChromosomes
Long strands of DNA packaged and Long strands of DNA packaged and compressed very tightlycompressed very tightly
Everyone has __23_________ copies Everyone has __23_________ copies of each chromosomeof each chromosome
1 pair of each of the 22 ‘autosomes’ <22>1 pair of each of the 22 ‘autosomes’ <22>• plus XX for a female (46XX) <1>plus XX for a female (46XX) <1>• or XY for a male (46XY) <OR 1>or XY for a male (46XY) <OR 1>
DIPLOID GENOMEDIPLOID GENOME
Chromosomes in Metaphase
Telomere
Telomere
Centromere
Short arm (p)
Long arm (q)
DNA (DeoxyriboNucleic Acid)DNA (DeoxyriboNucleic Acid)
2 major functions2 major functions Direction of all protein synthesis Direction of all protein synthesis Accurate transmission of this Accurate transmission of this
information from one generation to the information from one generation to the nextnext
Fundamental toFundamental to Cell metabolismCell metabolism Cell divisionCell division
DNA (DeoxyriboNucleic Acid)DNA (DeoxyriboNucleic Acid)
String of deoxyribose String of deoxyribose sugars joined by sugars joined by _____phosphate___________phosphate______ groups.groups.
Each sugar is attached Each sugar is attached to one of 4 possible to one of 4 possible nucleotide basesnucleotide bases
ADENINE (A), ADENINE (A), CYTOSINE (C ), CYTOSINE (C ), GUANINE (G) or GUANINE (G) or THYMINE (T)THYMINE (T)
Each hydrogen Each hydrogen bond hold two bond hold two DNA strands DNA strands together very together very tightly.tightly.
To form Watson-To form Watson-Crick base pairs, Crick base pairs, DNA strands must DNA strands must be anti parallel & be anti parallel & ____double ____double stranded_______stranded_______
DNA DNA
Double helix structureDouble helix structure 2 strands are held together by 2 strands are held together by
hydrogen bondshydrogen bonds 4 bases pairing rule4 bases pairing rule
o Adenine = Thymine Adenine = Thymine (A = T) (A = T) o Guanine = Cytosine Guanine = Cytosine (G = C)(G = C)
A G C G A T C T G G
DNA Base Pairing
Double helix consists of 2 complimentary strands of DNA.
T C G C T A G A C C
Some Definitions
Replicon: Unit in which replication occurs
Origin: Position at which replication initiates
Terminus: Position at which replication terminates
rep gene
Ope
rato
r si
teIter
ons
Dna
A bo
xes
AT-ric
h
RepliconOrigin
5’
3’
Conservative
Dispersive3
’
5’
DNA Replication is Semiconservative
Semi-conservative
Semiconservative Replication of Density-Semiconservative Replication of Density-Labeled DNALabeled DNA
DNA replication is semi-conservative: in the "next generation" molecule one strand is "old" and another is "new"
DNAunzips
AT
C
TA
G
TA
G
AT
C
DNA ReplicationSemi – conservative replication
A T CT A G
Original double strand DNA separates and
replicates2 new double strands
– each containing one parent
and one daughter strand
T A GA T C
A T C
T A G
Many enzymes are Many enzymes are involved in DNA involved in DNA
replicationreplication
The Enzymes of DNA The Enzymes of DNA ReplicationReplication
1. Topoisomerase - initiates the unwinding of the super 1. Topoisomerase - initiates the unwinding of the super coiled DNA by clipping the DNA backbone. coiled DNA by clipping the DNA backbone.
2. Helicase - separates the double strand by "melting" 2. Helicase - separates the double strand by "melting" the hydrogen bonds that hold the bases together. It the hydrogen bonds that hold the bases together. It requires energy (in the form of ATP ). requires energy (in the form of ATP ).
3. Primase - makes a short3. Primase - makes a short RNA RNA segment (called a segment (called a primer) that is complementary to the DNA strand at primer) that is complementary to the DNA strand at specific sites. It "primes" for DNA replication because specific sites. It "primes" for DNA replication because it provides an - OH for DNA polymerase to attach the it provides an - OH for DNA polymerase to attach the first DNA nucleotide. The RNA primer is later removed first DNA nucleotide. The RNA primer is later removed and the gap is filled in with DNA nucleotides. and the gap is filled in with DNA nucleotides.
4. DNA polymerase (III- major polymerase) - binds to 4. DNA polymerase (III- major polymerase) - binds to one side of the DNA and nucleotides to bond with their one side of the DNA and nucleotides to bond with their complementary pair complementary pair
5. Ligase – joins two large molecules via covalent 5. Ligase – joins two large molecules via covalent bonds-repair discontinued SS-DNA strandsbonds-repair discontinued SS-DNA strands
Common Features of Common Features of Replication OriginReplication Origin
Unique DNA sequence containing Unique DNA sequence containing multiple short repeated sequencemultiple short repeated sequence
Origins contain DNA sequences Origins contain DNA sequences recognized by replication initiator recognized by replication initiator proteinsproteins
AT-rich stretchAT-rich stretch
DNA replication starts within a special region of DNA called REPLICATION ORIGIN which is defined by a specific nucleotide sequence
Replication of DNA is ____semiconservative_____semiconservative_____________
Two Y-shaped replication forks are moving in the opposite directions during DNA replication
Initiation of DNA Initiation of DNA ReplicationReplication
DnaB is a DnaB is a helicase.helicase. An enzyme moves An enzyme moves
along DNA duplex along DNA duplex utilizing the utilizing the energy of ATP energy of ATP hydrolysis to hydrolysis to separate the separate the strands. strands.
5’-3’5’-3’ SSB: single SSB: single
strand binding strand binding protein.protein.
DNA synthesis always DNA synthesis always starts with a RNA starts with a RNA
primerprimer
The Role of RNA The Role of RNA Primer in DNA Primer in DNA
ReplicationReplication E. coliE. coli primase primase
catalyze the RNA catalyze the RNA Primer for DNA Primer for DNA SynthesisSynthesis dnaGdnaG
Primer: <15 ntsPrimer: <15 nts
DNA PolymeraseDNA Polymerase
Unable to separate the two strands Unable to separate the two strands of DNAof DNA
Only elongate a pre-existing DNA or Only elongate a pre-existing DNA or RNA (Primer)RNA (Primer)
Only add nucleotides to the 3’-Only add nucleotides to the 3’-hydorxyl group, i.e., only 5’-3’ hydorxyl group, i.e., only 5’-3’ synthesissynthesis
DNA Synthesis Occurs in the 5’3’ Direction
P P P P P PP PP
PP PP5’
3’ 5’
OH 3’
OH 3’
P P P P P PP PP
PP PP5’
3’ 5’
OH3’5’PPP
P P P P P PP PP
5’
3’ 5’
OH 3’PP PP P
PP
Incoming nuceolotidetriphosphate
Nucleotide monophosphateadded to chain with release
of diphosphate
DNA Synthesis is Semidiscontinous
5’3’5’3’
3’5’
Lagging strand synthesis
Leading strand synthesis
DNA Polymerase DNA Polymerase Exonuclease ActivityExonuclease Activity
The enzyme that synthesizes DNA is ______POLYMERASE__________________ self-correcting:: it has a proofreading activity
It is not trivial to replicate both DNA strands in the 5' to 3' direction!
Growing ForkGrowing Fork
Leading Leading strand: strand: continuouscontinuous
Lagging Lagging strand: strand: discontinudiscontinuousous
Lagging strand synthesisLagging strand synthesis
Leading Leading strand: strand: continuouscontinuous
Lagging Lagging strand: strand: discontinuousdiscontinuous
Okazaki Okazaki fragment: fragment:
Gap Gap RemovalRemoval
Pol IPol I 5’-3’ 5’-3’
exonucleaseexonuclease
Fills gapFills gap
LigaseLigase
DNA Pol I -exonuclease- reads the fragments and removes the RNA Primers.
LigaseLigase
DNA Ligase: adds phosphate in the remaining gaps of the phosphate - sugar backbone
Proofreading by 3’ to 5’ Proofreading by 3’ to 5’ ExonucleaseExonuclease
Proofreading by 3’ to 5’ Proofreading by 3’ to 5’ Exonuclease activity of Exonuclease activity of
DNA PolDNA Pol
Final Step-TerminationFinal Step-Termination
This process happens when the DNA This process happens when the DNA Polymerase (enzymes) reaches to an Polymerase (enzymes) reaches to an end of both strands. end of both strands.
The end of the parental strand The end of the parental strand where the last enzymes binds aren't where the last enzymes binds aren't replicated. replicated.
These ends of chromosomal DNA These ends of chromosomal DNA consists of noncoding DNA. consists of noncoding DNA.
From Genes to From Genes to ProteinsProteins
Patricia Caldani MSPatricia Caldani MS
DNA and RNA differ in 3 DNA and RNA differ in 3 waysways
RNARNA
Single-strandedSingle-stranded
Ribose (sugar)Ribose (sugar)
Uracil (base) Uracil (base) bonds to Adeninebonds to Adenine
DNADNA
Double-strandedDouble-stranded
Deoxiribose Deoxiribose (sugar)(sugar)
Thymine (base) Thymine (base) bonds to Adeninebonds to Adenine
The Genetic CodeThe Genetic Code
Every three bases of DNA is called Every three bases of DNA is called a ‘codon’a ‘codon’
Each codon specifies an amino Each codon specifies an amino acid acid
Codons specify amino acid Codons specify amino acid sequence of proteinsequence of protein
Alanine Ala A Leucine Leu L
Arginine Arg R Lysine Lys K
Asparagine Asn N Methionine Met M
Aspartic Acid Asp D Phenylalanine Phe F
Cysteine Cys C Proline Pro P
Glutamine Gln Q Serine Ser S
Glutamic Acid Glu E Threonine Thr T
Glycine Gly G Tryptophan Trp W
Histidine His H Tyrosine Tyr Y
I soleucine I le I Valine Val V
Amino Acid Code
•64 possible triplet codons
•Only 20 amino acids
•Code is “degenerate or redundant”
U URACIL C CYTOCINE A ADENINE G GUANINEU UUU Phe (F) CUU Leu (L) AUU Ile [I] GUU Val [V] U
UUC Phe (F) CUC Leu (L) AUC Ile [I] GUC Val [V] CUUA Leu (L) CUA Leu (L) AUA Ile [I] GUA Val [V] AUUG Leu (L) CUG Leu (L) AUG Met [M] GUG Val [V] G
C UCU Ser (S) CCU Pro [P] ACU Thr [T] GCU Ala [A] UUCC Ser (S) CCC Pro [P] ACC Thr [T] GCC Ala [A] CUCA Ser (S) CCA Pro [P] ACA Thr [T] GCA Ala [A] AUCG Ser (S) CCG Pro [P] ACG Thr [T] GCG Ala [A] G
A UAU Tyr [Y] CAU His [H] AAU Asn [N] GAU Asp [D] UUAC Tyr [Y] CAC His [H] AAC Asn [N] GAC Asp [D] CUAA Ter [end] CAA Gln [Q] AAA Lys [K] GAA Glu [E] AUAG Ter [end] CAG Gln [Q] AAG Lys [K] GAG Glu [E] G
G UGU Cys [C] CGU Arg [R] AGU Ser [S] GGU Gly [G] UUGC Cys [C] CGC Arg [R] AGC Ser [S] GGC Gly [G] CUGA Ter [end] CGA Arg [R] AGA Arg [R] GGA Gly [G] AUGG Trp [W] CGG Arg [R] AGG Arg [R] GGG Gly [G] G
Genetic Code
Exons Introns
Gene Structure
Promoter
UAAUAG ‘stop’UGA
AUGstart
Exon = coding sequenceIntron= intervening sequence (non-coding)
Protein Synthesis
DNA Code
Messenger - RNA
The Flow of InformationThe Flow of Information
DNA RNA Protein
transcription
translation
Protein Synthesis
The Flow of InformationThe Flow of Information
TranscriptionTranscription
Double DNA strands separateDouble DNA strands separate DNA sense strand acts as template DNA sense strand acts as template
and is ‘transcribed’ into messenger and is ‘transcribed’ into messenger RNA (mirror image of the DNA but RNA (mirror image of the DNA but Uracil instead of Thymine)Uracil instead of Thymine)
Introns are sliced out of the Introns are sliced out of the sequencesequenceDNA
mRNA
A T C G G
U A G C C
TranscriptionTranscription
This is the first step in Protein This is the first step in Protein Synthesis:Synthesis:
1. The instructions are 1. The instructions are ______________ ______________ (“transcribed”)(“transcribed”) to an RNA molecule. to an RNA molecule.
To sum up Transcription…To sum up Transcription… Info transferred from Info transferred from DNA to RNADNA to RNA
What is the Enzyme involved in What is the Enzyme involved in Transcription?Transcription?
Answer Answer RNA PolymeraseRNA Polymerase
Transcription has 3 Transcription has 3 steps…steps…
1 – RNA 1 – RNA Polymerase Polymerase binds to the binds to the gene’s gene’s promoterpromoter (DNA)(DNA) (like a (like a starting line starting line in a race).in a race).
2 – RNA Polymerase 2 – RNA Polymerase UNWINDSUNWINDS the the DNA molecule. The DNA molecule. The DNA DNA nucleotidesnucleotides are exposed. are exposed.
3 – 3 – RNA PolymeraseRNA Polymerase adds adds complimentary ______________complimentary ______________ to to separated DNA strand.separated DNA strand.
** Remember ** Remember RNA has RNA has ______________ ______________ instead of instead of ThymineThymine for for a base.a base.
The RNA Polymerase will continue The RNA Polymerase will continue transcription until it reaches the transcription until it reaches the “stop signal”“stop signal” on the DNA molecule on the DNA molecule (like a finish line).(like a finish line).
Then the RNA strand is released and Then the RNA strand is released and goes on to the next step…goes on to the next step…Translation Translation
Transcription
TranscriptionTranscription
TranscriptiTranscriptionon
intron
exon 1 2 3
1 2transcription
factors
RNApolymerase
5’ 3’
intron
exon 1 2 3
1 2transcription
factors
RNApolymerase
5’ 3’
TranscriptiTranscriptionon
intron
exon 1 2 3
1 2transcription
factors
5’GGATTCGTGCTGCTAA
5’GGAUUCGUGCUGCUAA
RNApolymerase
TranscriptiTranscriptionon
intron
exon 1 2 3
1 2transcription
factors
primarytranscript
RNApolymerase
TranscriptiTranscriptionon
promotorintron
exon 1 2 3
1 2
transcriptionfactors
AAAAAAAAAAmature mRNA
primarytranscript
RNA splicing
RNApolymerase
TranscriptiTranscriptionon
3 types of RNA3 types of RNA
mRNA (messenger RNA)mRNA (messenger RNA)
rRNA (ribosomal RNA)rRNA (ribosomal RNA)
tRNA (transfer RNA)tRNA (transfer RNA)
Messenger RNAMessenger RNA
Delivers Delivers ____________________________ to the site of to the site of Translation.Translation.
mRNA instructions are written in mRNA instructions are written in
““3-nucleotide” sequences3-nucleotide” sequences.. These sequences are called These sequences are called codonscodons.. Ex.Ex.
UUU, CUG, ACU, etc. UUU, CUG, ACU, etc. There are There are 6464 possible codons. possible codons.
TranslationTranslation
Remember what happens in Remember what happens in TranscriptionTranscription?? DNA to RNADNA to RNA
In In TranslationTranslation……RNARNA is coded for is coded for Amino AcidsAmino Acids..
TranslationTranslation
mRNA leaves the nucleus mRNA leaves the nucleus In the cytoplasm, ribosomes In the cytoplasm, ribosomes
attach to the mRNA ensuring the attach to the mRNA ensuring the correct amino acid, for each correct amino acid, for each codon, is added to a growing chain codon, is added to a growing chain of amino acids which forms the of amino acids which forms the resulting protein.resulting protein.
mRNA molecule
cap structure
polyadenylation site(AATAAA)
AAAAAAAAA
Ribosome
cytoplasm
5’
3’
TranslationTranslation
AAAAAAAAA
mRNA molecule
Ribosome
Peptidechain
cytoplasm
5’
3’
TranslationTranslation
Translation takes Translation takes place in the place in the ______________ ______________
tRNA (Transfer tRNA (Transfer RNA) molecules RNA) molecules carry single carry single amino acidsamino acids..
They also have They also have an an OPPOSITE “3-OPPOSITE “3-nucleotide” nucleotide” sequencesequence called called anticodonsanticodons..
rRNA (Ribosomal RNA) molecules rRNA (Ribosomal RNA) molecules are like assembly lines are like assembly lines they carry: they carry: 1 mRNA1 mRNA 2 tRNA2 tRNA
7 Steps in Translation7 Steps in Translation
1 – mRNA 1 – mRNA start start codoncodon starts the starts the process at the process at the P site.P site.
2 – the next 2 – the next tRNAtRNA bonds to the next bonds to the next codon at the codon at the A siteA site..
3 – A & P are holding 2 tRNA’s…a 3 – A & P are holding 2 tRNA’s…a ______________ ______________ bond is formed bond is formed between 2 amino acids.between 2 amino acids.
4 – 4 – tRNA detaches from P-site, leaves tRNA detaches from P-site, leaves behind amino acid, leaves Ribosomebehind amino acid, leaves Ribosome..
5 – tRNA at A-site moves to the P-site. 5 – tRNA at A-site moves to the P-site. Now a new codon is ready at the A-Now a new codon is ready at the A-site for another tRNA.site for another tRNA.
6 – tRNA detaches from P-site, leaves 6 – tRNA detaches from P-site, leaves behind amino acid, behind amino acid, ____________________________ ribosome.ribosome.
7 – 7 – (Steps 2 – 6 repeat until a stop (Steps 2 – 6 repeat until a stop codon is reached).codon is reached). Ex. UAG, UAA, Ex. UAG, UAA, UGA.UGA.
A A new proteinnew protein is then released into is then released into the cell.the cell.
Translation
Translation
The Human GenomeThe Human Genome Only ~5% of our DNA actually Only ~5% of our DNA actually
codes for proteins. Little variation codes for proteins. Little variation exists from person to person.The exists from person to person.The remainder is ‘junk’remainder is ‘junk’
‘‘Junk’ DNA includes repetitive Junk’ DNA includes repetitive sequences such as micro and sequences such as micro and minisatellites. Varies a lot between minisatellites. Varies a lot between individuals allowing ‘DNA individuals allowing ‘DNA fingerprinting’fingerprinting’
RNA polymerase responsible: transcription, and ribosome (which is responsible for translation) are very accurate enzymes and make very few mistakes. IF RNA polymerase or a ribosome make a mistake it is not highly detrimental to the cell
An alteration in the nucleotide An alteration in the nucleotide sequence of the gene is called a sequence of the gene is called a ________________________ .________________________ .
Mutations in the gene affect the Mutations in the gene affect the structure and the function of all the structure and the function of all the proteins expressed from the mutant proteins expressed from the mutant gene.gene.
MutationsMutations A change in the DNA sequence of A change in the DNA sequence of
the genethe geneo Germline mutation (inherited)– Germline mutation (inherited)–
present in every cell in the body present in every cell in the body o Somatic mutation (acquired) – Somatic mutation (acquired) –
present only in the descendants of present only in the descendants of that cell that cell
All cells acquire mutations as they All cells acquire mutations as they dividedivide
Mutations can alter protein product Mutations can alter protein product of DNA, stop gene working or of DNA, stop gene working or activate geneactivate gene
Types of Mutation Types of Mutation (in coding sequence)(in coding sequence)
AGC TAGC TGA CCC GGA CCC G Deletion Deletion
AGC TTC AGC TTC CCCCG ACC CGG ACC CG InsertionInsertion
AGC TTC AGC TTC TTC TTCTTC TTC GAC CCGGAC CCG ExpansionExpansion
ATC TATC TGGC GAC CCGC GAC CCG Point Point mutationmutation
ATC ATC TGATGA Nonsense Nonsense ‘stop’‘stop’
AGC TTC GAC CCG Wild type
Cells require mechanisms to ___________________ DNA damage
Mutation rate reflects the number of damaging events in DNA vs. the number of
corrections
2 broad types of changes:Single base mutationsStructural distortions
DNA Repair
Repair Mechanisms Direct repair, e.g., photoreactivation of thymine dimers by a light-dependent enzyme (photolyase)
Nucleotide excision repair Mechanism Multiple systems, some of which act generally, others more specifically e.g. glycosylases and AP endonucleases Found in bacteria, archaea and eucaryotes UV damage and repair of bulky lesions
Tolerance systems allow replication of damaged DNA but with higher error frequencies
Recombination repair uses homologous recombination to obtain the correct sequence from an undamaged source
Mismatch repair removes mismatches which arise during DNA replication
Base mutation/distortion
5’ and 3’ incisions
Exonuclease excision
Replacement strand synthesis
Ligase seals backbone nick
Nucleotide Excision Repair
Lectures 1-4 Overview DNA replication is semi-conservative: both
strands are used as templates for new strands
DNA synthesis occurs exclusively 5’3’
DNA synthesis is semi-discontinuous (except inrolling circle replication where the two strandsare replicated from independent origins):
5’3’5’3’
3’5’
Lagging strand synthesis
Leading strand synthesis
Leading strand synthesis is primed once with a primase-synthesized RNA primer
Lagging strand synthesis is primed repeatedly with Okazaki fragments
Summary
DNA damage can be of a variety of types
Cells require mechanisms to repair DNA damage
Different mechanisms exist
Nucleotide excision repair is a sequential and coordinated series of enzymatic events by which DNA damage cane be repaired
DNA polymerases have proof-reading activity whichcorrects incorporation of incorrect nucleotides
Proteins at the replication fork:
In eukaryotes replication is initiated at multiple ori on different chromosomes; termination is poorly understood
Lectures 1-4 Overview
DNA PolIIIPrimase
HelicaseSSBParental DNA
Leading strand
Lagging strand (Okazaki fragment)
5’3’
3’
5’3’5’
+ DNA PolI+ Ligase
Exercise 1.Each column in the table below represents three nucleotides. Within each column, fill in the cells that are blank by using information from the cell that is not blank.
DNA strand TAC GGG
mRNA UCG
CCU
Amino Acid Leu
Transcription and Translation problems
1. polypeptide: proline-tyrosine-histidine-valine-glutamic acidWhat is the base sequence of the mRNA that codes for the polypeptide? A. CCG-UAU-CAU-GUA-GAAB. GAA-GUA-CAU-UAA-CCGC. CCG-GUA-GAA-UAA-AUGD. CCG-GUA-CAU-CUA-CCG
2. Use a genetic code table to determine the polypeptide sequence synthesized from the mRNA below. Assume that translation begins at the first nucleotide at the 5' end.5' AUGAAGUGUUAACCC 3‘
3. What RNA sequence is transcribed by the gene with the DNA template strand 5’ TTGAGCGCGTA 3’
1. Major features of the Watson and Crick model of the double helix
2. How DNA is replicated in Eukaryotes and Prokaryotes3. The major steps involved in DNA replication4. How the process of DNA replication ensures accuracy5. Mutations and how they can be harmful or beneficial6. Scientists Watson Crick, Meselson and Stahl, and Chargaff
discoveries7. Chromosome structure and function8. The purpose of Transcription and the differences between
RNA and DNA9. How amino acids are designated in a stretch of RNA10. Steps involved in protein synthesis, their locations, and the
important players involved11. Be able to transcribe and translate DNA sequence