Lecture8

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