nucleic-acidslecture

8/2/2019 nucleic-acidsLECTURE

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NUCLEIC

ACIDS


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Topic Outline:

History of Nucleic Acids

Structure and Function

Types of Nucleic Acids

1. DNA

2. RNA

Central Dogma of Life


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Friedrich Miescher in

1869 isolated what he called nucleinfrom the

nuclei of pus cells


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Richard Altmann in 1889

Nuclein was shown to have acidicproperties, hence it became called nucleic

acid


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1920s

the tetranucleotide hypothesis wasintroduced


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The Tetranucleotide

hypothesis Up to 1940 researchers were convinced

that hydrolysis of nucleic acids yielded the

four bases in equal amounts. Nucleic acid was postulated to contain

one of each of the four nucleotides, thetetranucleotide hypothesis.

Takahashi (1932) proposed a structure ofnucleotide bases connected byphosphodiester linkages.


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The Tetranucleotide

hypothesis

adenine uracil

cytosine guaninephosphate

phosphatephosphate

phosphate

pentose

pentose pentose

pentose


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Astbury and Bell in 1938

First X-ray diffraction pattern of DNA ispublished.

The pattern indicates a helicalstructure, indicated periodicity.


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X-ray diffraction of DNA


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Wilkins & Franklin (1952): X-ray

crystallography


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Avery, MacLeod, and Mc

Carty in 1944 demonstrate DNA could transform

cells.

Supporters of the tetranucleotidehypothesis did not believe nucleic acidwas variable enough to be a molecule

of heredity and store geneticinformation.


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DNA is Genetic Material


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Erwin Chargaff in late

1940s used paper chromatography for

separation of DNA hydrolysates.

Amount of adenine is equal to amountof thymine and amount of guanine isequal to amount of cytosine.


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Hershey and Chase in 1952

confirm DNA is a molecule of heredity.


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The Hershey-Chase Experiment


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The Hershey-Chase Experiment


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Watson and Crick in 1953

determine the structure of DNA


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Watson & Crick Base pairing


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Francis Crick in 1958

proposes the central dogma of molecular biology .

Kornberg purifies DNA polymerase I


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1969

Entire genetic code determined


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Nucleic Acids

Nucleic Acids are very long, thread-like

polymers, made up of a linear array of monomers

called nucleotides.

Nucleic acids vary in size in nature

tRNA molecules contain as few as 80 nucleotides

Eukaryotic chromosomes contain as many as

100,000,000 nucleotides.


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Two types of nucleic acid

are found Deoxyribonucleic acid (DNA)

Ribonucleic acid (RNA)

d


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DNA and RNA

DNA

deoxyribonucleic acidnucleic acid that stores genetic informationfound in the nucleus of a mammalian cell.

RNAribonucleic acid3 types of RNA in a cell

Ribosomal RNAs (rRNA) are components of ribosomesMessenger RNAs (mRNA) carry genetic informationTransfer RNAs (tRNA) are adapter molecules in translation


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The distribution of nucleic

acids in the eukaryotic cell DNA is found in the nucleus

with small amounts in mitochondria and

chloroplasts RNA is found throughout the cell


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The nucleus contains the cells DNA

(genome)

Nucleus


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RNA is synthesized in the nucleus and

exported to the cytoplasm

Nucleus

Cytoplasm


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DNA as genetic material: The

circumstantial evidence1. Present in all cells and virtually restricted to the nucleus2. The amount of DNA in somatic cells (body cells) of any

given species is constant (like the number of

chromosomes)3. The DNA content of gametes (sex cells) is half that of

somatic cells.In cases of polyploidy (multiple sets of chromosomes)

the DNA content increases by a proportional factor4. The mutagenic effect of UV light peaks at 253.7nm. The

peak for the absorption of UV light by DNA


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NUCLEIC ACID STRUCTURE

Nucleic acids are polynucleotides

Their building blocks are nucleotides


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NUCLEOTIDE STRUCTURE

PHOSPATE SUGAR

Ribose orDeoxyribose

NUCLEOTIDE

BASE

PURINES PYRIMIDINESAdenine (A)Guanine(G) Cytocine (C)Thymine (T)

Uracil (U)

N l tid St t


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All nucleotides contain three components:

1. A nitrogen heterocyclic base2. A pentose sugar

3. A phosphate residue

Nucleotide Structure


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Ribose is a pentose

C1

C5

C4

C3 C2

O


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RIBOSE DEOXYRIBOSE

CH2OH

H

OH

C

C

OH OH

C

O

H HH

C

CH2

OH

H

OH

C

C

OH H

C

O

H HH

C

Spot the difference

Chemical Structure of DNA vs RNA


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Ribonucleotides have a 2-OH

Deoxyribonucleotides have a 2-H

Chemical Structure of DNA vs RNA


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THE SUGAR-PHOSPHATE

BACKBONE The nucleotides are all

orientated in the same

direction The phosphate group joins the

3rd Carbon of one sugar to the5th Carbon of the next in line.

P

P

P

P

P

P

P


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ADDING IN THE BASES

The bases areattached to the 1st

Carbon Their order is

importantIt determines thegenetic information ofthe molecule

P

P

P

P

P

P

G

C

C

A

T

T


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DNA IS MADE OF

TWO STRANDS OF

POLYNUCLEOTIDEP

P

P

P

P

P

C

G

G

T

A

A

P

P

P

P

P

P

G

C

C

A

T

T

Hydrogen bonds


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DNA IS MADE OF TWO STRANDS OF

POLYNUCLEOTIDE

The sister strands of the DNA molecule run in oppositedirections (antiparallel)

They are joined by the bases

Each base is paired with a specific partner:

A is always paired with T

G is always paired with C

Purine with Pyrimidine

The sister strands are complementary but not identical The bases are joined by hydrogen bonds, individually

weak but collectively strong

There are 10 base pairs per turn


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Structure of Nucleotide

Bases

Purines & Pyrimidines

5 End


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5 End

3 End

Nucleotides

are

linked byphosphodiest

er

bonds


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CENTRAL DOGMA

OF LIFE

From DNA to Protein


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DNA to Protein

DNA acts as a manager in the process ofmaking proteins

DNA is the template or starting sequencethat is copied into RNA that is then usedto make the protein


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Central Dogma

One gene one protein


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Central Dogma

This is the same for bacteria to humans

DNA is the genetic instruction or gene

DNA RNA is called Transcription RNA chain is called atranscript

RNA Protein is called Translation


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Expression of

Genes

Some genes aretranscribed in large

quantities becausewe need largeamount of thisprotein

Some genes aretranscribed insmall quantities

because we needonly a smallamount of thisprotein


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Nucleotides as Language

We must start to think of the nucleotides A,G, C and T as part of a special language thelanguage of genes that we will see translated

to the language of amino acids in proteins


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Genes as Information Transfer

A gene is the sequence of nucleotides withina portion of DNA that codes for a peptide or afunctional RNA

Sum of all genes = genome


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STEP 1 DNA REPLICATION


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DNA

Replication Semiconservative

Daughter DNA is adouble helix with 1

parent strand and 1 newstrand

Found that 1 strandserves as the templatefor new strand


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DNA Template

Each strand of the parent DNA is used as a templateto make the new daughter strand

DNA replication makes 2 new complete doublehelices each with 1 old and 1 new strand


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Replication Origin

Site where replicationbegins 1 in E. coli

1,000s in human

Strands are separated toallow replication machinerycontact with the DNA Many A-T base pairs because

easier to break 2 H-bonds that3 H-bonds

Note anti-parallel chains

R li ti F k


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Replication Fork

Bidirectional movement of the DNA replication machinery


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THE REPLICATION FACTORY

DNA replication is an intricate processrequiring the concerted action of manydifferent proteins.

The replication proteins are clusteredtogether in particular locations in the cell and

may therefore be regarded as a smallReplication Factory that manufactures DNAcopies.


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THE REPLICATION FACTORY

The DNA to be copied is fed through the factory,much as a reel of film is fed through a movieprojector.

The incoming DNA double helix is split into twosingle strands and each original single strand

becomes half of a new DNA double helix.Because each resulting DNA double helix retainsone strand of the original DNA, DNA replicationis said to be semi-conservative.


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DNA REPLICATION PROTEINS

DNA replication requires a variety of proteins.

Each protein performs a specific function inthe production of the new DNA strands.

Helicase, made of six proteins arranged in aring shape, unwinds the DNA double helixinto two individual strands.


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Single-strand binding proteins, or SSBs, aretetramers that coat the single-stranded DNA.

This prevents the DNA strands from reannealingto form double-stranded DNA.

Primase is an RNA polymerase that synthesizesthe short RNA primers needed to start the

strand replication process.


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DNA polymerase is a hand-shaped enzyme that stringsnucleotides together to form a DNA strand.

The sliding clamp is an accessory protein that helps hold theDNA polymerase onto the DNA strand during replication.

RNAse H removes the RNA primers that previously beganthe DNA strand synthesis.

DNA ligase links short stretches of DNA together to createone long continuous DNA strand.


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Components of the DNA

Replication

Polymerase & Proteins


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Polymerase & Proteins

Coordinated

One polymerase complex apparently synthesizesleading/lagging strands simultaneously

Even more complicated in eukaryotes


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STRAND SEPARATION

To begin the process of DNA replication, the two doublehelix strands are unwound and separated from eachother by the helicase enzyme.

The point where the DNA is separated into singlestrands, and where new DNA will be synthesized, isknown as the replication fork.

Single-strand binding proteins, or SSBs, quickly coat thenewly exposed single strands. SSBs maintain theseparated strands during DNA replication.

Replication Fork


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Replication Fork

Bidirectional movement of the DNA replication machinery


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STRAND SEPARATION

Without the SSBs, the complementary DNA strandscould easily snap back together.

SSBs bind loosely to the DNA, and are displaced whenthe polymerase enzymes begin synthesizing the newDNA strands.


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NEW STRAND SYNTHESIS

Now that they are separated, the two singleDNA strands can act as templates for theproduction of two new, complementary DNA

strands.

Remember that the double helix consists of

two antiparallel DNA strands withcomplementary 5 to 3 strands running inopposite directions.


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NEW STRAND SYNTHESIS

Polymerase enzymes can synthesize nucleicacid strands only in the 5 to 3 direction,hooking the 5 phosphate group of an

incoming nucleotide onto the 3 hydroxylgroup at the end of the growing nucleic acidchain.

Because the chain grows by extension off the3 hydroxyl group, strand synthesis is said to

proceed in a 5 to 3 direction.


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NEW STRAND SYNTHESIS Even when the strands are separated, however, DNA

polymerase cannot simply begin copying the DNA.

DNA polymerase can only extend a nucleic acid chain butcannot start one from scratch.

To give the DNA polymerase a place to start, an RNApolymerase called primase first copies a short stretch of theDNA strand.

This creates a complementary RNA segment, up to 60nucleotides long that is called a primer.


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NEW STRAND SYNTHESIS Now DNA polymerase can copy the DNA strand.

The DNA polymerase starts at the 3 end of the RNA primer,and, using the original DNA strand as a guide, begins tosynthesize a new complementary DNA strand.

Two polymerase enzymes are required, one for eachparental DNA strand.

Due to the antiparallel nature of the DNA strands, however,the polymerase enzymes on the two strands start to move inopposite directions.


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NEW STRAND SYNTHESIS One polymerase can remain on its DNA template

and copy the DNA in one continuous strand.

However, the other polymerase can only copy a

short stretch of DNA before it runs into the primerof the previously sequenced fragment.

It is therefore forced to repeatedly release the DNAstrand and slide further upstream to beginextension from another RNA primer.


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NEW STRAND SYNTHESIS The sliding clamp helps hold this DNA polymerase onto the

DNA as the DNA moves through the replication machinery.The sliding clamp makes the polymerase processive.

The continuously synthesized strand is known as the leading

strand, while the strand that is synthesized in short pieces isknown as the lagging strand.

The short stretches of DNA that make up the lagging strand

are known as Okazaki fragments.


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THE LAGGING STRAND

Before the lagging-strand DNA exits thereplication factory, its RNA primers must beremoved and the Okazaki fragments must be

joined together to create a continuous DNAstrand.

The first step is the removal of the RNAprimer.


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THE LAGGING STRAND

RNAse H, which recognizes RNA-DNA hybrid helices,degrades the RNA by hydrolyzing its phosphodiesterbonds. Next, the sequence gap created by RNAse H isthen filled in by DNA polymerase which extends the 3end of the neighboring Okazaki fragment.

Finally, the Okazaki fragments are joined together byDNA ligase that hooks together the 3 end of onefragment to the 5 phosphate group of the neighboringfragment in an ATP- or NAD+-dependent reaction.


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REPLICATION IN ACTION

The process begins when the helicaseenzyme unwinds the double helix to exposetwo single DNA strands and create two

replication forks.

DNA replication takes place simultaneouslyat each fork. The mechanism of replication is

identical at each fork.

How is DNA Synthesized?


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Original theory

Begin adding nucleotides at origin

Add subsequent bases following pairing rules

Expect both strands to be synthesized simultaneously

This is NOT how it is accomplished



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Actually how DNA is synthesized Simple addition of nucleotides along one strand, as

expected

Called the leading strand

DNA polymerase reads 35 along the leadingstrand from the RNA primer

Synthesis proceeds 53 with respect to the newdaughter strand

Remember how the nucleotides are added!!!!!

53


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Mi t k d i


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Mistakes during

Replication Base pairing rules must be maintained Mistake = genome mutation, may have consequence

on daughter cells

Only correct pairings fit in the polymerase active

site If wrong nucleotide is included

Polymerase uses its proofreading ability to cleave thephosphodiester bond of improper nucleotide Activity 35

And then adds correct nucleotide and proceeds downthe chain again in the 5 3 direction


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Proofreading


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DNA Repair

For the rare mutations occurring duringreplication that isnt caught by DNApolymerase proofreading

For mutations occurring with daily assault

If no repair

In germ (sex) cells inherited diseases

In somatic (regular) cells cancer


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CONSEQUENCES OF GENETIC ERRORS

:SOURCES OF GENETIC VARIATION

Mutation - any novel genetic change in thegene complement or genotype relative to the

parental genotypes, beyond that achieved by

genetic recombination during meiosis.

Mutations are changes in DNA structure, and

therefore changes in protein and phenotype.


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CONSEQUENCES OF GENETIC ERRORS

SOURCES OF GENETIC VARIATION

Mutations are rare! For every 100 millionnucleotides added to a developing DNA strand

only one mistake occurs on average.

Mutations are heritable; and may be

beneficial, neutral, lethal, detrimental or

harmful to the organism.


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Types of Mutation

1. Induced

viruses, UV radiation, some chemicals(nitric acid changes cytosine to uracil) ormutagens (or carcinogens - benzene,cigarette smoke).

i


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Types of Mutation

2. Spontaneous

Proofreading mistakes during DNA replication(Base substitutions) - not necessarily a serious

change.

Frame shift mutation (Addition or deletion of a

base) - serious change!

Types of Mutation


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Types of Mutation

A 3 letter code or codon is analogous to three letter words in asentence.

Original sequence

THE CAT SAW THE DOG

Base or letter substitutions

THE BAT SAW THE DOG

THE CAT SAW THE HOGTHE CAB SAW THE DOG

THE CAT SAW SHE DOG

THE CAT SAD THE DOG

THE CAT SAW THE DOC

Types of Mutation


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Types of Mutation

Deletions

THE CAT SAW TED OG

THE ATS AWT HED OG

Additions

THE CAT SAW THE ZDO G

THE CMA TAS WTH EDO G

f i


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Types of Mutation

3. Jumping genes, transposable elements, ortransposons.

Discovered by Barbara McClintok (1956)

while studying color variation in Indiancorn.

Won Nobel prize in 1983.

T f M t ti


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Types of Mutation

3. Jumping genes, transposable elements, ortransposons.

Patches of yellow sometimes occur among the purplegrains of Indian corn. She explain this by assumingthat the gene was being interrupted by a foreignsequence of DNA.

These foreign bits of DNA could insert or removethemselves from a stretch of DNA causing the genesthat they affected to be turned on or off. Such"jumping genes" could copy themselves and moveabout within the genome of the organism theyoccupied.

T f M t ti


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Types of Mutation

4. Chromosomal mutations (disruption in chromosomalmorphology - inversions and translocations).

5. Homeotic genes master genes that regulate suites of other genes and

may affect developmental pathways especially duringembryogenesis. Mutations in these master genes can

cause genetic anomalies. For example, a fruit fly thatpossesses legs where antennae should be, or amosquito that has its mouth parts transformed intolegs.

Effect of Mutation


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Effect of Mutation

Uncorrected Replication


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Uncorrected Replication

Errors

Mismatch repair Enzyme complex recognizes mistake and excises newly-

synthesized strand and fills in the correct pairing

Mismatch Repair contd


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Mismatch Repair cont d

Eukaryotes labelthe daughter strandwith nicks to

recognize the newstrand

Separates new fromold

Ch i l M difi ti


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Chemical Modifications

Thymine Dimers


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Thymine Dimers

Caused by exposure to UV light

2 adjacent thymine residues becomecovalently linked


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Repair

Mechanisms Different enzymes

recognize, excisedifferent mistakes

DNA polymerasesynthesizes properstrand

DNA ligase joins newfragment with thepolymer


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STEP 2 - TRANSCRIPTION

Transcription


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Transcription

The region of the double-stranded DNAcorresponding to a specific gene is copiedinto an RNA molecule, called messenger RNA(mRNA).

RNA differs from DNA Ribose is the sugar rather than deoxyribose

ribonucleotides

U instead of T; A, G and C the same

Single stranded Can fold into a variety of shapes that allows RNA to

have structural and catalytic functions

RNA Differences


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RNA Differences

RNA Differences


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RNA Differences

Transcription


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Transcription

Similarities to DNA replication Open and unwind a portion of the DNA 1 strand of the DNA acts as a template

Complementary base-pairing with DNA

Differences RNA strand does not stay paired with DNA

DNA re-coils and RNA is single stranded

RNA is shorter than DNA

RNA is several 1000 bp or shorter whereas DNA is250 million bp long

RNA P l Catalyzes the formation


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RNA Polymerase Catalyzes the formationof the phosphodiesterbonds between thenucleotides (sugar tophosphate)

Uncoils the DNA, addsthe nucleotide one at a

time in the 5 to 3 fashion

Uses the energy trappedin the nucleotidesthemselves to form the

new bonds

Template to Transcripts


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Template to Transcripts

The RNA transcript is identical to the NON-template strand with the exception of the Tsbecoming Us

RNA Elongation


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RNA Elongation

Reads template 3to 5

Adds nucleotides5 to 3 (5

phosphate to 3hydroxyl)

Synthesis is thesame as the

leading strand ofDNA

Differences in


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Differences in

DNA and RNA Polymerases

RNA polymerase adds ribonucleotides notdeoxynucleotides

RNA polymerase does not have the ability toproofread what they transcribe

RNA polymerase can work without a primer

RNA will have an error 1 in every 10,000nucleotides (DNA is 1 in 100,000,000 nucleotides)

Types of RNA


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Types of RNA

messenger RNA (mRNA) codes for proteins

ribosomal RNA (rRNA) forms the core of theribosomes, machinery for making proteins

transfer RNA (tRNA) matches code foramino acid on mRNA and positions the rightamino acid in place during protein synthesis

How does the process of


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How does the process of

transcription begin?

The DNA serves as the template forproducing an RNA transcript or copy ofinformation stored on the DNA molecule.

The DNA molecule must open up and allowan enzyme called RNA polymerase read and

connect together the sequence of nucleotidesin the proper order.


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STEP 3 TRANSLATION

RNA to Protein


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RNA to Protein

Translation is the process of turningmRNA into protein

Translate from one language (mRNA

nucleotides) to a second language(amino acids)

Genetic code nucleotide sequencethat is translated to amino acids of theprotein

DNA Code


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Nucleotides read 3 at a time meaning that thereare 64 combinations for a codon (set of 3nucleotides)

Only 20 amino acids More than 1 codon per AA degenerate code with the

exception of Met and Trp (least abundant AAs inproteins)


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Reading Frames

Translation can occur in 1 of 3 possible readingframes, dependent on where decoding starts in themRNA

Transfer RNA Translation requires an


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Molecules Translation requires an

adaptor molecule that

recognizes the codon onmRNA and at a distantsite carries theappropriate amino acid

Intra-strand base pairingallows for thischaracteristic shape

Anticodon is oppositefrom where the aminoacid is attached


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Wobble Base Pairing

Due to degenerate code for amino acids sometRNA can recognize several codons because the 3rdspot can wobble or be mismatched

Allows for there only being 31 tRNA for the 61codons

Attachment of AA to tRNA


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Attachment of AA to tRNA

Aminoacyl-tRNA synthase is the enzymeresponsible for linking the amino acid to thetRNA

A specific enzyme for each amino acid andnot for the tRNA

2 Adaptors Translate


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p

Genetic Code to Protein

1

2

Ribosomes Complex machinery thatt l t i th i


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controls protein synthesis

2 subunits

1 large catalyzes the peptidebond formation

1 small binds mRNA and tRNA

Contains protein and RNA rRNA central to the catalytic

activity

Folded structure is highly conserved

Protein has less homology andmay not be as important

Ribosome Structures


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May be free in cytoplasm or attached to the ER

Subunits made in the nucleus in the nucleolus andtransported to the cytoplasm

Ribosomal Subunits


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1 large subunit catalyzes the formation of the peptide bond 1 small subunit matches the tRNA to the mRNA

Moves along the mRNA adding amino acids to growing proteinchain

Ribosomal Movement


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4 binding sites

mRNA binding site

Peptidyl-tRNA binding site (P-site)

Holds tRNA attached to growing end of the peptide

Aminoacyl-tRNA binding site (A-site)

Holds the incoming AA

Exit site (E-site)

E-site

Summary


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Summary

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