Download ppt - Cytogenetics 2 replication, transcription and translation

Dr. SAHAR ABO ELFADL 1

الرحمن الله بسمالرحيم

DNA DNA ReplicationReplication, ,

TranscriptionTranscription & & TranslationTranslation

Dr. SAHAR ABO ELFADL 22007-2008

DNA Replication


• In the late 1950s, three different mechanisms were proposed for the replication of DNA– Conservative model

• Both parental strands stay together after DNA replication

– Semi-conservative model• The double-stranded DNA contains one parental and one

daughter strand following replication

– Dispersive model• Parental and daughter DNA are interspersed in both strands

following replication

Proposed Models of DNA ReplicationProposed Models of DNA Replication


Three models for DNA replication

The most accepted


Directionality of DNA

• You need to number the carbons!– it matters!

OH

CH2

O

4

5

3 2

1

PO4

N base

ribose

nucleotide

This will beIMPORTANT!!


The DNA backbone

• Putting the DNA backbone together– refer to the 3 and 5 ends

of the DNA• the last trailing carbon

OH

O

3

PO4

base

CH2

O

base

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

5

Sounds trivial, but…this will be

IMPORTANT!!


Anti-parallel strands

• Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons

– DNA molecule has “direction”– complementary strand runs in

opposite direction

3

5

5

3


Bonding in DNA

….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?

3

5 3

5

covalentphosphodiester

bonds

hydrogenbonds


Copying DNA

• Replication of DNA– base pairing allows

each strand to serve as a template for a new strand

– new strand is 1/2 parent template & 1/2 new DNA (semi-conservative).


DNA Replication

• Large team of enzymes coordinates replication

Let’s meetthe team…


Replication: 1st step

• Unwind DNA– helicase enzyme

• unwinds part of DNA helix• stabilized by single-stranded binding proteins

single-stranded binding proteins replication fork

helicase


DNAPolymerase III

Replication: 2nd step

But…We’re missing

something!What?

Where’s theENERGY

for the bonding!

Build daughter DNA strand add new

complementary bases DNA polymerase III


energy

ATPGTPTTPATP

Energy of Replication

Where does energy for bonding usually come from?

ADPAMPGMPTMPAMPmodified nucleotide

We comewith our own

energy!

And weleave behind a

nucleotide!

Youremember

ATP!Are there other ways

to get energyout of it?


Limits of DNA polymerase III can only build onto FREE 3

end of an existing DNA strand

Leading & Lagging strands

5

5

5

5

3

3

3

53

53 3

Leading strand

Lagging strand

Okazaki fragments

ligase

Okazaki

Leading strand continuous synthesis

Lagging strand Okazaki fragments joined by ligase

“spot welder” enzyme

DNA polymerase III

3

5

growing replication fork


DNA polymerase III

Replication fork / Replication bubble

5

3 5

3

leading strand

lagging strand

leading strand

lagging strandleading strand

5

3

3

5

5

3

5

3

5

3 5

3



5

5

5

5

53

3

5

5lagging strand

5 3


DNA polymerase III

RNA primer built by primase serves as starter sequence

for DNA polymerase III

Limits of DNA polymerase III can only build onto 3 end of

an existing DNA strand

Starting DNA synthesis: RNA primers

5

5

5

3

3

3

5

3 53 5 3


primase

RNA


DNA polymerase I removes sections of RNA

primer and replaces with DNA nucleotides

But DNA polymerase I still can only build onto 3 end of an existing DNA strand

Replacing RNA primers with DNA

5

5

5

5

3

3

3

3


DNA polymerase I

RNA

ligase


Loss of bases at 5 ends in every replication chromosomes get shorter with each replication limit to number of cell divisions?

DNA polymerase III

All DNA polymerases can only add to 3 end of an existing DNA strand

Chromosome erosion

5

5

5

5

3

3

3

3


DNA polymerase I

RNA

Houston, we have a problem!


Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions

Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells

high in stem cells & cancers -- Why?

telomerase

Telomeres

5

5

5

5

3

3

3

3


TTAAGGGTTAAGGGTTAAGGG


Replication fork

3’

5’

3’

5’

5’

3’

3’ 5’

helicase

direction of replication

SSB = single-stranded binding proteins

primase

DNA polymerase III

DNA polymerase III

DNA polymerase I

ligase

Okazaki fragments

leading strand

lagging strand

SSB


Fast & accurate!

Human cell • copies its 6 billion

bases• Completes mitosis in

only few hours• remarkably accurate• only ~1 error per 100

million bases• ~30 errors per cell cycle


NOW

Let us see together this video about

DNA REPLICATION

C:\Program Files\Real\RealPlayer\realplay.exe


DNA Replication

• Origins of replicationOrigins of replication

1. Replication ForksReplication Forks: hundredshundreds of Y-Y-shapedshaped regions of replicating DNA replicating DNA moleculesmolecules where new strands are growing.

ReplicationReplicationForkFork

Parental DNA MoleculeParental DNA Molecule

3’

5’

3’

5’

http://www.ultranet.com/~jkimball/BiologyPages/D/DNAReplication.html


DNA Replication

• Origins of replicationOrigins of replication

2. Replication BubblesReplication Bubbles:

a. HundredsHundreds of replicating bubbles (Eukaryotes)(Eukaryotes).

b. SingleSingle replication fork (bacteria).(bacteria).

Bubbles Bubbles

http://faculty-staff.ou.edu/S/Barbara.Safiejko-Mroczk-1/3113/Lectures/Lecture%2013/13.dna.replicat.html


DNA ReplicationDNA Replication

• Strand SeparationStrand Separation:

1.1. HelicaseHelicase: enzyme which catalyze the unwindingunwinding and separationseparation (breaking

H- Bonds) of the parental double helix.

2.2. Single-Strand Binding ProteinsSingle-Strand Binding Proteins: proteins which attach and help keep the

separated strands apart.



• Priming:Priming:

1.1. RNA primersRNA primers: before new DNA strands can form, there must be small pre-existing

primers (RNA)primers (RNA) present to start the addition of new nucleotides (DNA Polymerase)(DNA Polymerase).

2.2. PrimasePrimase: enzyme that polymerizes (synthesizes) the RNA Primer.



• Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:

1.1. DNA PolymeraseDNA Polymerase: with a RNA primerRNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the synthesis of a new DNA strand in the

5’ 5’ to 3’ directionto 3’ direction.

RNARNAPrimerPrimerDNA PolymeraseDNA Polymerase

NucleotideNucleotide

5’

5’ 3’



2.2. Leading StrandLeading Strand: synthesized as a single polymersingle polymer in the 5’ to 3’ direction5’ to 3’ direction.

RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides

3’5’

5’



3.3. Lagging StrandLagging Strand: also synthesized in the 5’ to 3’ direction5’ to 3’ direction, but

discontinuouslydiscontinuously against overall direction of replication.

RNA PrimerRNA Primer

Leading StrandLeading Strand

DNA PolymeraseDNA Polymerase

5’

5’

3’

3’

Lagging StrandLagging Strand

5’

5’

3’

3’



4.4. Okazaki FragmentsOkazaki Fragments: series of short segments on the lagging strand.lagging strand.

Lagging Strand

RNARNAPrimerPrimer

DNADNAPolymerasePolymerase

3’

3’

5’

5’

Okazaki FragmentOkazaki Fragment


DNA ReplicationDNA Replication5.5. DNA ligaseDNA ligase: a linking enzyme that

catalyzes the formation of a covalent bond from the 3’ to 5’ end3’ to 5’ end of joining stands.

Example: joining two Okazaki fragments together.Example: joining two Okazaki fragments together.

Lagging Strand

Okazaki Fragment 2Okazaki Fragment 2

DNA ligaseDNA ligase

Okazaki Fragment 1Okazaki Fragment 1

5’

5’

3’

3’

Dr. SAHAR ABO ELFADL 322007-2008

DNA Transcription

& Translation


The Link Between DNA and Protein

• DNA contains the molecular blueprint of every cell

• Proteins are the “molecular workers” of the cell• Proteins control cell shape, function,

reproduction, and synthesis of biomolecules• The information in DNA genes must therefore

be linked to the proteins that run the cell


Transcription• Process by which

genetic information encoded in DNA is copied onto messenger RNA

• Occurs in the nucleus• DNA mRNA

Translation• Process by which

information encoded in mRNA is used to assemble a protein at a ribosome

• Occurs on a Ribosome

• mRNA protein


11 22

catalytic site

tRNA docking sites

Attached amino acidtRNAtransfer

Smallsubunit

rRNAribosomal

Largesubunit

CC

AA

GG

AA

UU

GG

GG

AA

GG

UU

UU

AA

UU

GG

GG

mRNAmRNAmessengermessenger

AA GG UU

Met

anticodon

Three Types of RNA


Transcription and Translation

• DNA directs protein synthesis in a two-step process

1. Information in a DNA gene is copied into mRNA in the process of transcription

2. mRNA, together with tRNA, amino acids, and a ribosome, synthesize a protein in the process of translation


Information Flow:

DNA

RNA

Protein


The Genetic Code• The base sequence in a DNA gene

dictates the sequence and type of amino acids in translation

• Bases in mRNA are read by the ribosome in triplets called codons

• Each codon specifies a unique amino acid in the genetic code

• Each mRNA also has a start and a stop codon



Overview of Transcription

• Transcription of a DNA gene into RNA has three stages

– Initiation

– Elongation

– Termination


Initiation

• Initiation phase of transcription1. DNA molecule is unwound and strands are

separated at the beginning of the gene sequence

2. RNA polymerase binds to promoter region at beginning of a gene on template strand



Elongation

1. RNA polymerase synthesizes a sequence of RNA nucleotides along DNA template strand

2. Bases in newly synthesized RNA strand are complementary to the DNA template strand

3. RNA strand peels away from DNA template strand as DNA strands repair and wind up



Elongation

• As elongation proceeds, one end of the RNA drifts away from the DNA; RNA polymerase keeps the other end temporarily attached to the DNA template strand



Termination

– RNA polymerase reaches a termination sequence and releases completed RNA strand




mRNA

– The DNA is in the nucleus and the ribosomes are in the cytoplasm

– The genes that encode the proteins for a biochemical pathway are not clustered together on the same chromosome

Each gene consists of multiple segments of DNA that encode for protein, called exons

Exons are interrupted by other segments that are not translated, called introns


IntronsIntrons

snipped out

snipped out

Introns

Introns

snipped out

snipped out

exonsexonsDNADNA

intronsintronspromoterpromoter

Transcription from DNA to RNATranscription from DNA to RNAInitialInitial

transcripttranscript

SplicingSplicing

completedcompletedmRNA transcriptmRNA transcript


mRNA

– Transcription of a gene produces a very long RNA strand that contains introns and exons

– Enzymes in the nucleus cut out the introns and splice together the exons to make true mRNA

– mRNA exits the nucleus through a membrane pore and associates with a ribosome


Ribosomes

• Ribosomes are large complexes of proteins and rRNA


Ribosomes

• Ribosomes are composed of two subunits

– Small subunit has binding sites for mRNA and a tRNA

– Large subunit has binding sites for two tRNA molecules and catalytic site for peptide bond formation


Transfer RNAs

• Transfer RNAs hook up to and bring amino acids to the ribosome

• There is at least one type of tRNA assigned to carry each of the twenty different amino acids

• Each tRNA has three exposed bases called an anticodon

• The bases of the tRNA anticodon pair with an mRNA codon within a ribosome binding site


Translation

• Ribosomes, tRNA, and mRNA cooperate in protein synthesis, which begins with initiation:

1. The mRNA binds to the small ribosomal subunit

2. The mRNA slides through the subunit until the first AUG (start codon) is exposed in the first tRNA binding site…


Translation

3. The first tRNA carrying methionine (and anticodon UAC) binds to the mRNA start codon completing the initiation complex

4. The large ribosomal subunit joins the complex


A tRNA with an A tRNA with an attached methionine attached methionine amino acid binds to a amino acid binds to a small ribosomal small ribosomal subunit, forming an subunit, forming an initiation complex. initiation complex.

Translation:Initiation (1)


The initiation The initiation complex binds to complex binds to end of mRNA and end of mRNA and travels down until it travels down until it encounters an AUG encounters an AUG codon in the mRNA. codon in the mRNA.

The anticodon of the The anticodon of the tRNA in the initiation tRNA in the initiation complex forms base complex forms base pairs with the AUG pairs with the AUG codon.codon.



The large The large ribosomal subunit ribosomal subunit binds to the small binds to the small subunit, with the subunit, with the mRNA between the mRNA between the two subunits.two subunits.

The methionine The methionine tRNA is in the first tRNA is in the first tRNA site on the tRNA site on the large subunit. large subunit.



The second tRNA enters the The second tRNA enters the second tRNA site on the large second tRNA site on the large ribosomal subunit.ribosomal subunit.

Which tRNA binds depends Which tRNA binds depends on the ability of its anticodon on the ability of its anticodon (CAA in this example) to base (CAA in this example) to base pair with the codon (GUU in pair with the codon (GUU in this example) in the mRNA.this example) in the mRNA.

tRNAs with a CAA anticodon tRNAs with a CAA anticodon carry an attached valine carry an attached valine amino acid, which was added amino acid, which was added to it by enzymes in the to it by enzymes in the cytoplasm.cytoplasm.

Translation:Elongation 1


The "empty" tRNA is The "empty" tRNA is released and the ribosome released and the ribosome moves down the mRNA, moves down the mRNA, one codon to the right.one codon to the right.

The tRNA that is attached The tRNA that is attached to the two amino acids is to the two amino acids is now in the first tRNA now in the first tRNA binding site and the binding site and the second tRNA binding site second tRNA binding site is empty.is empty.



The catalytic site on The catalytic site on the large subunit the large subunit catalyzes the catalyzes the formation of a peptide formation of a peptide bond linking the amino bond linking the amino acids methionine to acids methionine to valine.valine.

The two amino acids The two amino acids are now attached to are now attached to the tRNA in the the tRNA in the second binding second binding position.position.



Another tRNA enters the Another tRNA enters the second tRNA binding site second tRNA binding site carrying its attached carrying its attached amino acid.amino acid.

The tRNA has an The tRNA has an anticodon that pairs with anticodon that pairs with the codon. (Here, the CAU the codon. (Here, the CAU mRNA codon pairs with a mRNA codon pairs with a GUA tRNA anticodon.)GUA tRNA anticodon.)

The tRNA molecule carries The tRNA molecule carries the amino acid histidine the amino acid histidine (his).(his).




Binding of tRNAs, & Binding of tRNAs, & formation of peptide formation of peptide bonds continues.bonds continues.

Ribosome reaches Ribosome reaches STOP codon (UAG).STOP codon (UAG).

Protein "release Protein "release factors" signal the factors" signal the ribosome to release ribosome to release the protein.the protein.

The mRNA is also The mRNA is also released and large & released and large & small subunits small subunits separate.separate.


The catalytic site forms The catalytic site forms a new peptide bond, in a new peptide bond, in this example, between this example, between the valine and the the valine and the histidine.histidine.

A three-amino acid A three-amino acid chain is now attached chain is now attached to the tRNA in the to the tRNA in the second tRNA binding second tRNA binding site.site.

The empty tRNA in the The empty tRNA in the first site is released first site is released and the ribosome and the ribosome moves one codon to moves one codon to the right.the right.

Translation:Termination


GG GG GG AA GG CC GG AA UU UU UU

CC AA AA CC AA UU CC CC UU

Methionine Glycine Valine etc.

GG GG GG AA GG TT TT CC TT GG AA

templateDNA strand

(a) complementaryDNA strand

(b) mRNA

(c) tRNA

(d) protein

amino acids

anticodons

codons

gene

etc.

etc.

etc.

etc.

GG TT CC CC CC CC AA AA AA TT CC

Complementary Base Pairing


MOVI TIME


Effects of Mutations on Proteins

• Recall that mutations are changes in the base sequence of DNA

• Most mutations are categorized as– Substitutions– Deletions– Insertions– Inversions – Translocations



• Inversions and translocations– When pieces of DNA are broken apart

and reattached in different orientation or location

– Not problematic if entire gene is moved – If gene is split in two it will no longer code

for a complete, functional protein



• Insertions or deletions– Nucleotides are added or subtracted from a

gene– Reading frame of RNA codons is changed

• THEDOGSAWTHECAT is changed by deletion of the letter “S” to THEDOGAWTHECAT

– Resultant protein has very different amino acid sequence; almost always is non-functional



• Nucleotide substitutions (point mutations)– An incorrect nucleotide takes the place of a

correct one– Protein structure and function is unchanged

because many amino acids are encoded by multiple codons

– Protein may have amino acid changes that are unimportant to function (neutral mutations)



• Effects of nucleotide substitutions– Protein function is changed by an altered

amino acid sequence (as in gly val in hemoglobin in sickle cell anemia)

– Protein function is destroyed because DNA mutation creates a premature stop codon



Mutations Fuel Evolution

• Mutations are heritable changes in the DNA• Approx. 1 in 105-106 eggs or sperm carry a

mutation• Most mutations are harmful or neutral• Mutations create new gene sequences and are

the ultimate source of genetic variation• Mutant gene sequences that are beneficial may

spread through a population and become common


How Are Genes Regulated?

• The human genome contains ~ 30,000 genes• A given cell “expresses” (transcribes) only a

small number of genes• Some genes are expressed in all cells• Other genes are expressed only

– In certain types of cells– At certain times in an organism’s life– Under specific environmental conditions


The End