Genetic Engineering l3

8/3/2019 Genetic Engineering l3

http://slidepdf.com/reader/full/genetic-engineering-l3 1/151

Principles & techniques of Genetic Engineering



Genetic Engineering Principles & Techniques:

A : Structure and Function of The Genetic Material : Lectures 3 & 4 •Genetic Engineering – Alteration of Genome and its manipulationthat involves molecular biological techniques.•Molecular Biology stands on the principle of Central Dogma :

Replication, Transcription Translation

DNA RNA PROTEINReverse Transcriptase

•Structure of the Genetic material, DNA, in Prokaryotes & Eukaryotes•Functions : DNA Replication , in Prokaryotes & Eukaryotes•Structure of the Genetic material, RNA, in Prokaryotes & Eukaryotes•Functions : DNA Transcription, in Prokaryotes & Eukaryotes



B : Structure and Function of The Genetic Material:Gene Expression : Lectures 5 & 6

•DNA Replication, Transcription & Translation: in Prokaryotes and•Eukaryotes ( DNA Synthesis, RNA Synthesis, Protein synthesis)

C. Experimental Gene Manipulation: Lectures 7 & 8 Genetic Engineering Techniques:• Gene cloning: Molecular Techniques: rDNA Technology• DNA, RNA and Protein Extraction/ Isolation• Restriction Digestion, Purification,• Cloning vehicles, Ligation, Transformation and Screening,• Transfection & Selection, Clone stabilization• PCR and its applications• DNA Synthesis, Oligonucleotide and primers

D. Application of Genetic Engineering / rDNA Techniques :Biotechnology: Lectures 9 & 10 • Research, Medicine, Agriculture& GM foods.



Basics of Molecular biology



Basic differences between eukaryotes and

prokaryotesAttribute Eukaryotes Prokaryotes

Organisms Plants, animals and fungi bacteria and cyanobacteria

Cell wall No (animals); Yes (plants) yesChromosomesegregation

Mitotic spindle Cell membrane

meiosis + _Ribosome size 80 s 70 sCell organelleNuclearmembrane

+ Absent

Endoplasmicreticulum

+ -

Golgi apparatus + -

Mitochondria + -Chloroplast + -



Molecular biology: definition• Molecular biology is the study of molecular underpinnings of theprocess of replication, transcription and translation of the genetic

material.



Components involve in molecular biology

DNA

RNA

Protein



Gene : Unit of heredity• The DNA segments that carries

genetic information are calledgenes.• It is normally a stretch of DNA

that codes for a type of proteinor for an RNA chain that has afunction in the organism.

• Genes hold the information to

build and maintain anorganism's cells and passgenetic traits to offspring.



Deoxyribonucleic acid(DNA)

• DNA is a nucleic acid that contains the geneticinstructions used in the development and functioningof all known living organisms and some viruses.

• DNA is a set of blueprints needed to construct othercomponents of cells, such as proteins and RNAmolecules.



• Two long strands makes theshape of a double helix.• two strands run in opposite

directions to each other andare therefore anti-parallel.• Chemically, DNA consists of

two long polymers of simpleunits called nucleotides, withbackbones made of base,

sugars and phosphategroups.Fig : DNA double helix



• The DNA double helix is stabilized by hydrogen bonds between thebases attached to the two strands.

• One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugarribose in RNA.

Ribose



• The DNA chain is 22 to 26 Ångströms wide(2.2 to 2.6 nanometres), and one nucleotideunit is 3.3 Å (0.33 nm) long.

Size:



• DNA is a nucleic acid, made of longchains of nucleotides

DNA and RNA are polymers of

nucleotides

Figure 10.2A

Nucleotide

Phosphategroup

Nitrogenousbase

Sugar

Polynucleotide Sugar-phosphate backbone

DNA nucleotide

Phosphate

group

Nitrogenous base(A, G, C, or T)

Thymine (T)

Sugar(deoxyribose)



• DNA has four kinds of bases, A, T, C, and G

Figure 10.2B

Pyrimidines

Thymine (T) Cytosine (C)

Purines

Adenine (A) Guanine (G)



• James Watson and Francis Crick workedout the three-dimensional structure of

DNA, based on work by Rosalind Franklin

DNA is a double-stranded helix



• Hydrogen bonds between bases holdthe strands together: A and T, C and G

Figure 10.3D

Ribbon model Partial chemical structure Computer model

Hydrogen bond



• The sequence of codons in DNA spellsout the primary structure of apolypeptide

– Polypeptides form proteins that cells and organismsuse

Review: The flow of genetic information in

the cell is DNA—RNA---protein



• Mutations are changes in theDNA base sequence

– caused by errors in DNA replication or by mutagens

– change of a single DNA nucleotide causes sickle-celldisease

Mutations can change the meaning of

genes



Normal hemoglobin DNA

mRNA

Normal hemoglobin

Glu

Mutant hemoglobin DNA

mRNA

Sickle-cell hemoglobin

Val



• Types of mutations

mRNA

NORMAL GENE

BASE SUBSTITUTION

BASE DELETION

Protein Met Lys Phe Gly Ala

Met Lys Phe Ser Ala

Met Lys Leu Ala His

Missing



Deletion

Duplication

Inversion

Homologouschromosomes

Reciprocaltranslocation

Nonhomologouschromosomes

•Chromosomal changes can be large or small



DNA, RNA, protein overview



DNA, RNA, protein overview

Questions about the genome in an organism:

How much DNA, how many nucleotides?

How many genes are there?

What types of proteins appear to be coded by these genes?

Questions about the proteome:

What proteins are present?

Where are they?

When are they present - underwhat conditions?



Linking nucleotides5’

3’

3’

3’

3’

3’

5’

3’

3’

What next?

3’ 3’

3’

3’

2 n m

Hydrogen bonds

N-H------N

N-H------O

Adenine

Guanine

Thymine

Cytosine

Linking nucleotides:

The 3’-OH of one nucleotide islinked to the 5’-phosphate of the next nucleotide



Base pairing5’

3’

3’

3’

3’

3’

5’

3’

3’

3’

3’

3’

G

C

T

A

T

A

G

C

T

A

Base pairing (Watson-Crick):

A/T (2 hydrogen bonds)

G/C (3 hydrogen bonds)

Always pairing a purine and apyrimidine yields a constant width

DNA base composition:

A + G = T + C (Chargaff’s rule)

DNA

RNA

Mutations

Amino acids,

protein structure



5’ 3’

DNA conventions

1. DNA is a right-handed helix

2. The 5’ end is to the left by convention

-ATCGCAATCAGCTAGGTT- sense (forward)

antisense (reverse)-TAGCGTTAGTCGATCCAA-3’ 5’

5’ -ATCGCAATCAGCTAGGTT- 3’

3’ -TAGCGTTAGTCGATCCAA- 5’



DNA StructureSome more facts:1. Forces stabilizing DNA structure: Watson-Crick-H-bonding and base stacking

(planar aromatic bases overlap geometrically and electronically → energy gain)

2. Genomic DNAs are large molecules:Eschericia coli : 4.7 x 10 6 bp; ~ 1 mm contour lengthHuman: 3.2 x 10 9 bp; ~ 1 m contour length

3. Some DNA molecules (plasmids) are circular and have no free ends:

mtDNAbacterial DNA (only one circular chromosome)

4. Average gene of 1000 bp can code for average protein of about 330 amino acids

5. Percentage of non-coding DNA varies greatly among organisms

Organism # Base pairs # Genes Non-coding DNA

small virus 4 x 103

3 very little ‘typical’ virus 3 x 10 5 200 verylittlebacterium 5 x 10 6 3000 10 - 20%yeast 1 x 10 7 6000 > 50%human 3.2 x 10 9 30,000? 99%

amphibians < 80 x 10 9 ? ?

plants < 900 x 10 9 23,000 - >50,000 > 99%



Reading framesReading frame (also open reading frame):

The stretch of triplet sequence of DNA that potentially encodesa protein. The reading frame is designated by the initiation orstart codon and is terminated by a stop codon.

Position 1 CAG AUG AGG UCA GGC AUAgln met arg ser gly ile

Position 2 C AGA UGA GGU CAG GCA UA

arg trp gly gln ala

Position 3 CA GAU GAG GUC AGG CAU Aasp glu val arg his

- a reading frame is not always easily recognizable- each strand of RNA/DNA has three possible starting

points (position one, two, or three):

- mutations within an open reading frame that delete or add nucleotides can disruptthe reading frame ( frameshift mutation ):

Wildtype CAG AUG AG G UCA GGC AUA GAG

gln met arg ser gly ile glu

Mutant CAG AUG AG U CAG GCA UAG AG

gln met ser gln ala

Up to 30% of mutationscausing humane diseaseare due to prematuretermination of translation(nonsense mutations or

frameshift)



MutationsMutation:

Sources of mutation:

Spontaneous mutations: mutations occur for unknown reasons

Induced mutations: exposure to substance (mutagen) known to cause mutations,e.g. X-rays, UV light, free radicals

Mutations may influence one or several base pairs

a) Nucleotide substitutions (point mutation)1) Transitions (Pu ↔ Pu; Py ↔ Py)2) Transversions (Pu ↔ Py)

In-class exerciseHow many transition and transversionevents are possible?

2 transitions: T ↔ C; A ↔ G4 transversions: T ↔ A; T ↔ G

C ↔ A; C ↔ G

b) Insertion or deletion (“indels”)

- one to many bases can be involved- frequently associated with repeated sequences (“hot spots”)- lead to frameshift in protein-coding genes, except when N = 3X- also caused by insertion of transposable elements into genes

“Weighting” of mutation events plays important role for phylogenetic analyses(model of sequence evolution)

any heritable change in DNA



MutationsMutations may influence phenotype

a) Silent (or synonymous) substitution

- nucleotide substitution without amino acid change- no effect on phenotype

- mostly third codon position- other possible silent substitutions: changes in non-coding DNA

b) Replacement substitution

- causes amino acid change- neutral : protein still functions normally- missense : protein loses some functions (e.g. sickle cell anemia: mutation in ß-globin)

c) Sense/nonsense substitution

- sense: involves a change from a termination codonto one that codes for an amino acid

- nonsense: creates premature termination codon

Mutation rates= a measure of the frequency of a given mutation per generation

- mutation rates are usually given for specific loci (e.g. sickle cell anemia)- the rate of nucleotide substitutions in humans is on the order of 1 per 100,000,000- range varies from 1 in 10,000 to 1 in 10,000,000,000- every human has about 30 new mutations involving nucleotide substitutions- mutation rate is about twice as high in male as in female meiosis



MutationsA single amino acid substitution in a protein causes sickle-cell disease



What is Genome ?

• Genomeis the entirety of an organism's hereditaryinformation.

• It is encoded either in DNA or, for many types of virus, in RNA.

• The genome includes both the genes and the non-

coding sequences of the DNA.



organism Size (bp) genenumber average gene density chromosome

number

Homo sapiens (human) 3.2 billion ~25,000 1 gene /100,000 bases 46

Mus musculus (mouse) 2.6 billion ~25,000 1 gene /100,000 bases 40

Drosophila melanogaster (fruit fly)

137 million 13,000 1 gene / 9,000 bases 8

Arabidopsis thaliana (plant) 100 million 25,000 1 gene / 4000 bases 10Caenorhabditis elegans (roundworm) 97 million 19,000 1 gene / 5000 bases 12

Saccharomyces cerevisiae (yeast)

12.1 million 6000 1 gene / 2000 bases 32

Escherichia coli (bacteria) 4.6 million 3200 1 gene / 1400 bases 1

H. influenzae (bacteria) 1.8 million 1700 1 gene /1000 bases 1

comparative genome sizes of organisms



Why Genome analysis ?•The prediction of genes in uncharacterised genomicsequences.

•To obtain the complete sequences of as many genomesas possible.

•For Genetic modification.

•Genetic modification to develop new varieties at afaster rate like BT cotton and BT brinjal.



Replication, Transcription, andTranslation

• Before a cell can divide, the DNA inthe nucleus of the cell must beduplicated.

• Since the DNA molecule consists of two complimentary stands, if thosetwo strands separate and the right

conditions are present, two newstands that are the compliments of the originals will be produced.

• Each new DNA molecule willconsist of one old stand, and a newcomplimentary strand.

• The gray strands in the figure tothe right are new strands in theprocess of being assembled.



Untwisting and replication of DNA

• each strand is a template for a new strand

helicase

DNA polymerase



Assembling the New Bases• The term semiconservative replication means that in the

new DNA molecule there is one old and one new strand.

• This is seen in the figure below.



DNA Replication• Since the DNA molecule is very large, there must be a way to copy it faster

than just unwinding from one end to the other!

• This happens when the DNA molecule separates at many sites, formingthousands of replication bubbles. This allows parts of the DNA message to bereplicated simultaneously in many locations.

• DNA polymerase adds new nucleotides , while DNA ligase joints the DNAsegments together.



Why the fuss about DNA replication??

• The process of DNA replication involves a number of enzymes and proteins, and it a bit more complicated than

seen in the previous slide.

• The important idea is that an exact duplication of the DNAmessage is required, so that each new cell in the body hasthe same set of genetic instructions as the cells thatpreceded it.

• This also insures that every new generation of individualshas the same genetic information as his/her parents.



• DNA replication begins at many specific sites

How can entire chromosomes be replicated during S phase

Figure 10.5A

Parental strandOrigin of replication

Bubble

Two daughter DNA molecules

Daughter strand



• Each strand of thedouble helix isoriented in the

opposite direction

Figure 10.5B

5 ′′′′ end 3 ′′′′ end

3 ′′′′ end 5 ′′′′ end

P

P

P

P

P

P

P

P

l3 ′′′′



• DNA

polymerase works inonly onedirection

5 ′′′′ end

P

P

Parental DNA

DNA polymerasemolecule

5 ′′′′

3 ′′′′

5 ′′′′

3 ′′′′

5 ′′′′

Daughter strand

synthesizedcontinuously

Daughterstrandsynthesizedin pieces

DNA ligase

Overall direction of replication

5 ′′′′

3 ′′′′

• Telomere

sequences are lost with each

replication.

• Cancer,a in

telomeres

DNA replication



DNA replication

• DNA replication, the basis for biological inheritance,is a fundamental process occurring in all livingorganisms to copy their DNA.

• In the process of "replication" each strand of theoriginal double-stranded DNA molecule serves astemplate for the reproduction of the complementarystrand.

• Two identical DNA molecules have been producedfrom a single double-stranded DNA molecule.



• In a cell, DNA replication begins at specific locations in the genome,called "origins".

• Unwinding of DNA at the origin, and synthesis of new strands, forms areplication fork.

• In addition to DNA polymerase, the enzyme that synthesizes the newDNA by adding nucleotides matched to the template strand, a numberof other proteins are associated with the fork and assist in the initiationand continuation of DNA synthesis.

• Cellular proofreading that ensure near perfect fidelity for DNAreplication.

The Replication of DNA




THE CHEMISTRY OF DNA SYNTHESIS




The Replication of DNATHE CHEMISTRY OF DNA SYNTHESIS

DNA synthesis requires dNTPs and a primer:template junction

DNA is synthesized by extending the 3’end of the prime



y y g pHydrolysis of pyrophosphate is the driving force forDNA Synthesis

G= -7 kcal/mole

DNA polymerases are processive enzymes; thus the rate



The thumb helps to maintain astrong association between theDNA polymerase and its substrate

p y p yof DNA synthesis is dramatically increased (~1000 bp/se

Exonucleases proofread newly synthesized DNA



THE REPLICATION FORK



THE REPLICATION FORKBoth strands of DNA are synthesized together at thereplication fork

DNA synthesis proceeds in a5’ to 3’direction



The structure of a DNA replication fork.

Because both daughter DNA strands are polymerized in the 5'to-3' direction, the DNA

synthesized on the lagging strand must be made initially as a series of short DNA molecules,called Okazaki fragments

DNA synthesis proceeds in a5 to 3 directionand issemi-discontinuous.

The initiation of a new strand of DNA requires an RNA



The initiation of a new strand of DNA requires an RNA

primer

A Special Nucleotide-Polymerizing Enzyme Synthesizes



RNA primer synthesis.Aschematic view of the reactioncatalyzed by DNA primase , theenzyme that synthesizes the

short RNA primers made onthe lagging strand using DNAas a template. Unlike DNApolymerase, this enzyme canstart a new polynucleotide

chain by joining two or threenucleoside triphosphatestogether . The primasesynthesizes a shortpolynucleotide in the 5'-to-3'

direction and then stops,making the 3' end of thisprimer available for the DNApolymerase

p y g y yShort RNA Primer Molecules on the Lagging Strand

RNA primers must be removed



RNA primers must be removed

to complete DNA replication

DNA helicasesunwind the double helix in advance of li ti f k



replication fork

Topoisomerasesremove supercoils produced by DNA



Topoisomerasesremove supercoils produced by DNA

unwinding at the replication fork

Replication fork enzymes extend the range of DNA



Replication fork enzymes extend the range of DNA

polymerase



THE SPECIALIZATION OF DNA POLYMERASES



DNA polymerases are specialized for different roles in the cell

INITIATION OF DNA REPLICATIONS ifi g i DNA di t th i iti ti



Specific genomic DNA sequences direct the initiationof DNA Replication

The replicon model of replication initiation

Replicator sequences (origin of replication) include



p q ( g p )

initiator binding sites and easily unwound DNA

Eukaryotic chromosomes are replicated exactly once percell cycle



cell cycleIncomplete replication causes chromosome breakage

FINISHING REPLICATION



Type II topoisomerases are requiredto separate daughter DNA molecules

Lagging-strand synthesis is unable to copy the extremeends of linear chromosomes



ends of linear chromosomes

One solution of the end problem is to use protein priming



Telomerase Replicates the Ends of EukaryoticChromosomes

h f l



The structure of telomerase.

The telomerase is a protein–RNA complex that carries an

RNA template for synthesizing

a repeating, G-rich telomereDNA sequence. Only the part of

the telomerase proteinhomologous to reverse

transcriptase is shown here(green). A reverse transcriptaseis a special form of polymerase

enzyme that uses an RNAtemplate to make a DNA strand;

telomerase is unique in carryingits own RNA template with it at

all times.

Telomerase solves the end problem by extending the 3end of the chromosome



Telomeres form a looped structure in the cell



RNARibonucleic Acid

• RNA is also a nucleic acid



– different sugar– U instead of T

– Single strand, usually

Phosphategroup

Nitrogenous base(A, G, C, or U)

Uracil (U)

Sugar(ribose)



Structure of RNASingle strandedRibose Sugar5 carbon sugar

Phosphate groupAdenine, Uracil, Cytosine, Guanine



Types of RNAThree main typesMessenger RNA (mRNA) – transfers DNAcode to ribosomes for translation.

Transfer RNA (tRNA) – brings aminoacids to ribosomes for protein synthesis.Ribosomal RNA (rRNA) – Ribosomes aremade of rRNA and protein.

Table 14.2Types of RNA

Type of RNA Functions in Function



Type of RNA Functions in Function

Messenger RNA(mRNA)

Nucleus,migratesto ribosomesin cytoplasm

Carries DNAsequenceinformation toribosomes

Transfer RNA(tRNA)

Cytoplasm Provides linkagebetween mRNAand amino acids;transfers aminoacids to ribosomes

Ribosomal RNA(rRNA)

Cytoplasm Structuralcomponentof ribosomes

Ribonucleic acid (RNA)



• RNA is a biologically important type of moleculethat consists of a long chain of nucleotide units.

• Each nucleotide consists of a nitrogenous base, aribose sugar, and a phosphate.

Double-stranded RNA



• Double-stranded RNA

(dsRNA) is RNA with twocomplementary strands,similar to the DNA foundin all cells.

• dsRNA forms the geneticmaterial of some viruses(double-stranded RNAviruses).

Types of RNAType Abbr Function Distribution



Messenger RNA mRNA Codes for protein All organisms

Ribosomal RNA rRNA Translation All organisms

Transfer RNA tRNA Translation All organisms

in post-transcriptional modification

Small nuclear RNA snRNA Splicing and otherfunctions

Eukaryotes andarchaea

Y RNARNA processing, DNAreplication Animals

Telomerase RNA Telomere synthesis Most eukaryotes

Antisense RNA aRNATranscriptional attenuation / mRNA degradation / mRNAstabilisation / Translation block

All organisms

Regulatory RNAs

• mRNA carries information about a protein sequence to the

Messenger RNA



mRNA carries information about a protein sequence to theribosomes, the protein synthesis factories in the cell.

• It is coded so that every three nucleotides (a codon)correspond to one amino acid.

• In eukaryotic cells, once precursor mRNA (pre-mRNA) hasbeen transcribed from DNA, it is processed to mature mRNA.This removes its introns—non-coding sections of the pre-mRNA.



• The mRNA is then exported from thenucleus to the cytoplasm, where it is boundto ribosomes and translated into its

corresponding protein form with the help of tRNA.

• In prokaryotic cells, which do not havenucleus and cytoplasm compartments,

mRNA can bind to ribosomes while it isbeing transcribed from DNA.

Transfer RNA



• Transfer RNA (tRNA) is a small RNA chain of about 80nucleotides that transfers a specific amino acid to agrowing polypeptide chain at the ribosomal site of proteinsynthesis during translation.

• It has sites for amino acid attachment and an anticodonregion for codon recognition• that site binds to a specific sequence on the messenger RNA

chain through hydrogen bonding.

Ribosomal RNA



• Ribosomal RNA (rRNA) is the catalyticcomponent of the ribosomes.• Eukaryotic ribosomes contain four different

rRNA molecules: 18S, 5.8S, 28S and 5SrRNA.

• rRNA molecules are synthesized in thenucleolus.

• In the cytoplasm, ribosomal RNA andprotein combine to form a nucleoproteincalled a ribosome.

• The ribosome binds mRNA and carries outprotein synthesis. Several ribosomes may beattached to a single mRNA at any time.

• rRNA is extremely abundant and makes up80% of the 10 mg/ml RNA found in a typicaleukaryotic cytoplasm.

Difference between RNA & DNA



RNA DNARNA nucleotides containribose sugar

DNA contains deoxyribose

RNA has the base uracil DNA has the base thyminepresence of a hydroxyl groupat the 2' position of the ribosesugar.

Lacks of a hydroxyl group atthe 2' position of the ribosesugar.

RNA is usually single-stranded

DNA is usually double-stranded



The Genetic CodeThis is the language of mRNA.Based on the 4 bases of mRNA.“Words” are 3 RNA sequences calledcodons.The strand aaacguucgccc would beseparated as aaa-cgu-ucg-ccc the amino

acids would then be Lysine – Arginine –Serine - Proline



Genetic Codes

Second Base

Virtually all organisms share the same genetic code“unity of life”



U C A G

U

C

A

G

GACU

GACU

GACU

GACU

U U UU U CU U AU U G

C U UC U CC U AC U G

AU UAU CAU AAU G

G U UG U CG U AG U G

phe

leu

leu

ile

met (start)

val

U C UU C CU C AU C G

C C UC C CC C AC C G

AC UAC CAC AAC G

G C UG C CG C AG C G

ser

pro

thr

ala

U AUU ACU AAU AG

C AUC ACC AAC AG

AAUAAC

AAGAAA

G AUG ACG AAG AG

tyr

stopstop

his

gln

asn

lys

asp

glu

U G UU G CU G AU G G

C G UC G CC G AC G G

AG UAG CAG AAG G

G G UG G CG G AG G G

cys

stoptrp

arg

ser

arg

gly

F i r

s t B

a s e

Th

Phenylalanine

UUU

Leucine

UUA

Serine

UCU

Tyrosine

UAU

Stop

UAA

Cysteine

UGU

Stop

UGA

Tryptophan

UGG



UUC UUG UCCUCAUCG

UAC UAGUGA

UGC

Leucine

CUUCUCCUACUG

Proline

CCUCCCCCACCG

Histidine

CAUCAC

Glutamine

CAACAG

Arginine

CGUCGCCGACGG

IsoleucineAUUAUCAUA

MethionineAUG

ThreonineACUACCACAACG

AsparagineAAUAAC

LysineAAAAAG

SerineAGUAGC

ArginineAGGAGA

Valine

GUUGUCGUAGUG

Alanine

GCUGCCGCAGCG

Aspartic Acid

GAUGAC

Glutamic Acid

GAAGAG

Glycine

GGUGGCGGAGGG

RNA and Protein Synthesis



A. The Structure of RNA

B. Types of RNAC. TranscriptionD. RNA Editing

E. The Genetic CodeF. TranslationG. The Roles of RNA and DNAH. Genes and Proteins

Concept Map



from to to make up

also called which functions to also called also called which functions towhich functions to

can be

RNA

Messenger RNA Ribosomal RNA Transfer RNA

mRNA Carry instructions rRNACombine

with proteins tRNABring

amino acids toribosome

DNA Ribosome Ribosomes

RNA

RNA structure3 major types of RNA



r ibo n ucleic a cid

4 bases

A =U =

C =

G =

AdenineUracil

Cytosine

Guanine

Pyrimidine (C 4N2H4 ) Purine (C 5 N4H4 )

Nucleoside Nucleotide

base

H HH H

OHOH

CH2O

OH

+ sugar (ribose)

4’5’

3’ 2’

1’

base + sugar + phosphate

O

sugar

P OO-

O-

PO 4--

RNA 3 major types of RNAmessenger RNA (mRNA); template for protein synthesistransfer RNA (tRNA); adaptor molecules that decode the genetic coderibosomal RNA (rRNA); catalyzing the synthesis of proteins

Thymine (DNA) Uracil (RNA)

Base interactions in RNA



Base pairing:

U/A/(T) (2 hydrogen bonds)

G/C (3 hydrogen bonds)

RNA base composition:

A + G = U + C/ Chargaff’s rule does not apply (RNA usually prevails as single strand)

RNA structure:

- usually single stranded

- many self-complementary regions → RNA commonly exhibits an intricate secondary structure

(relatively short, double helical segments alternated with single stranded regions)- complex tertiary interactions fold the RNA in its final three dimensional form

- the folded RNA molecule is stabilized by interactions (e.g. hydrogen bonds and base stacking)

RNA structure



Primary structure

Secondary structure

A

A) single stranded regions

C) hairpinC

DD) internal loop

EE) bulge loop

FF) junctionB

B) duplex

G

G) pseudoknot

formed by unpaired nucleotides

double helical RNA (A-form with 11 bp per turn)

duplex bridged by a loop of unpaired nucleotides

nucleotides not forming Watson-Crick base pairs

unpaired nucleotides in one strand,other strand has contiguous base pairing

three or more duplexes separated by singlestranded regions

tertiary interaction between bases of hairpin loop

and outside bases

RNA structureP i



Primary structure

Secondary structure

A

C

D

E

FB

G

Tertiary structure

RNA structureHow to predict RNA secondary/tertiary structure?



ow to p ed ct seco da y/te t a y st uctu e?Probing RNA structure experimentally:

- physical methods (single crystal X-ray diffraction, electron microscopy)

- chemical and enzymatic methods

- mutational analysis (introduction of specific mutations to test change in some

function or protein-RNA interaction)Thermodynamic prediction of RNA structure:

- RNA molecules comply to the laws of thermodynamics, therefore it should bepossible to deduce RNA structure from its sequence by finding the conformationwith the lowest free energy

- Pros: only one sequence required; no difficult experiments; does not rely onalignments

- Cons: thermodynamic data experimentally determined, but not always accurate;possible interactions of RNA with solvent, ions, and proteins

Comparative determination of RNA structure:

- basic assumption: secondary structure of a functional RNA will be conserved in theevolution of the molecule (at least more conserved than the primary structure);when a set of homologous sequences has a certain structure in common, this structure canbe deduced by comparing the structures possible from their sequences

- Pros: very powerful in finding secondary structure, relatively easy to use, only sequencesrequired, not affected by interactions of the RNA and other molecules

- Cons: large number of sequences to study preferred, structure constrains in fully conserved

regions cannot be inferred, extremely variable regions cause problems with alignment



RNA ANDRNA AND

PROTEIN SYNTHESISPROTEIN SYNTHESIS

DNA carries information that can be used to construct theproteins which form structures and regulate the body’s

activitiesi h i i l i i d



activities.• Protein synthesis involves two processes: transcription andtranslation.

• In transcription the DNA message is converted into anRNA molecule.

• In translation the RNA message is used to assemble aminoacids into a protein chain.

HowHow does our celldoes our cell makes verymakes veryi t t t ii t t t i



important proteinsimportant proteins• The production (synthesis) of proteinsproteins.•• 3 phases3 phases:

1.1. TranscriptionTranscription2.2. RNA processingRNA processing3.3. TranslationTranslation

•• DNADNA------RNARNA ------ProteinProtein

Before making proteins,Before making proteins, OurOur cellcellt fi t k RNAt fi t k RNA



must first make RNAmust first make RNA•• Question:Question:• How does RNARNA (ribonucleic acid)(ribonucleic acid)differ

from DNA (deoxyribonucleic acid)DNA (deoxyribonucleic acid)?

RNARNAdiff f DNADNA



RNARNAdiffers fromDNADNA1. RNARNAhas a sugar ribosesugar ribose

DNADNAhas a sugarsugar deoxyribosedeoxyribose

2. RNARNAcontains uraciluracil (U)(U)DNADNAhas thymine (T)thymine (T)

3. RNARNAmolecule is singlesingle--strandedstrandedDNADNAis doubledouble--strandedstranded



mRNA



mRNA• Carries instructions from DNA to the rest

of the ribosome.• Tells the ribosome what kind of protein to

make

• Acts like an email from the principal tothe cafeteria lady.

A Messenger RNA (mRNA)A Messenger RNA (mRNA)



A. Messenger RNA (mRNA)A. Messenger RNA (mRNA)

methionine glycine serine isoleucine glycine alanine stopcodon

proteinprotein

A U G G G C U C C A U C G G C G C A U A AmRNAmRNA

startcodon

Primary structure of a proteinPrimary structure of a proteinaa1 aa2 aa3 aa4 aa5 aa6

peptide bonds

codon 2 codon 3 codon 4 codon 5 codon 6 codon 7codon 1

If the cell is a school…If the cell is a school…



•• The Nucleus is the school officeThe Nucleus is the school office•• The Nucleolus is the principal’sThe Nucleolus is the principal’sofficeoffice

•• The DNA is the principalThe DNA is the principal•• RibosomesRibosomes are the cafeteria ladiesare the cafeteria ladies•• mRNA is the email from themRNA is the email from the

principal to the cafeteria ladyprincipal to the cafeteria lady

rRNA



rRNA• Part of the structure of a ribosome• Helps in protein production

tRNAA go-getter.Gets the right parts to make the right proteinaccording to mRNA instructions

RNARNA ProcessingProcessing



RNARNA ProcessingProcessing•• IntronsIntronsare pulled out andexonsexonscome

together.• End product is amature RNA moleculemature RNA molecule

that leaves thenucleusnucleusto thecytoplasm.cytoplasm.

•• IntronsIntrons bad……bad…… ExonsExons good!good!

RNARNA ProcessingProcessing



RNARNA ProcessingProcessingpre-RNA molecule

intron intronexon exon exon

exon exon exon

Mature RNA moleculeMature RNA molecule

exon exon exon

intron intron

splicesome splicesome

Cleaning up the Message



• When the genetic message iscopied to make mRNA, themessage contains unwantedbase sequences.

• The ‘junk’ sequences (calledintrons) are removed from themessage and the remaining

sequences (exons) are linkedtogether to produce a sequenceof codons that will translate intoa polypeptide.

• This process occurs before themessage leaves the nucleus.

Transcription



TranscriptionRNA molecules are produced by copying part of

the nucleotide sequence of DNA intocomplementary sequence in RNA, a processcalled transcription.

During transcription, RNA polymerase binds toDNA and separates the DNA strands. RNApolymerase then uses one strand of DNA as a

template from which nucleotides are assembledinto a strand of mRNA.

mRNA How Does it Work?



RNA Polymerase looks for a region on theDNA known as apromoter, where it bindsand begins transcription.RNA strands are then edited. Some partsare removed (introns) - which are notexpressed – and other that are left are

calledexonsor expressed genes.

– The DNA is transcribed into RNA, which

• The information constituting anorganism’s genotype is carried in its

sequence of bases



The DNA is transcribed into RNA, whichis translated into the polypeptide

DNA

RNA

Protein

TRANSCRIPTION

TRANSLATION

Prokaryotic gene expression



In prokaryotes, RNA polymerase binds to the -10 and -35 regions of the promoter relative to the start site of

transcription (+1)



promoter operator



Reverse transcription

• Reverse transcribing viruses replicate their genomes by



g p g yreverse transcribing DNA copies from their RNA;

• These DNA copies are then transcribed to new RNA.• Retrotransposans also spread by copying DNA and RNA

from one another.



Transcription

• Transcription, is the process of creating an equivalent RNAcopy of a sequence of DNA.



py q• Transcription is the first step leading togene expression.

• DNA RNA.

• During transcription, a DNA sequence is read by RNApolymerase, which produces a complementary, antiparallelRNA strand.

• Transcription results in an RNA complement that includesuracil (U) instead of thymine (T).

transcription

reverse transcription

• The stretch of DNA transcribed into an RNA molecule iscalled a transcriptionunit and encodes at least one gene

Transcription process



called a transcriptionunit and encodes at least one gene.• If the gene transcribed encodes for a protein, the result of

transcription is messenger RNA (mRNA).• This mRNA will be used to create that protein via the

process of translation.• Alternatively, the transcribed gene may encode for either

rRNA or tRNA, other components of the protein-assemblyprocess, or other ribozymes.

• A DNA transcription unit encoding for protein (the coding

sequence ) and regulatory sequences that direct and regulatethe synthesis of that protein.

• DNA is read from 3'→ 5' during

transcription.• the complementary RNA is created



t e co p e e ta y s c eatedfrom the 5'→ 3' direction.

• only one of the two DNA strands,called the template strand, is usedfor transcription because RNA isonly single-stranded.

• The other DNA strand is called thecoding strand.

Transcription produces genetic messagesin the form of mRNA



RNApolymerase

RNA nucleotide

Direction oftranscription

Newly made RNA

Templatestrand of DNA

• In transcription,DNA helix unzips

RNA l id

RNA polymerase

DNA of gene

PromoterDNA TerminatorDNA

Initiation



– RNA nucleotidesline up along onestrand of DNA,following the base-pairing rules

– single-strandedmessenger RNA

peels away andDNA strandsrejoin

Elongation

Termination

Area shownin Figure 10.9A

GrowingRNA

RNApolymerase

Completed RNA

Figure 10.9B

RNA transcripts of DNA



• Noncodingsegments

Eukaryotic RNA is processedbefore leaving the nucleus

DNAExon Intron IntronExon Exon

T i i



segments,introns, arespliced out

• A cap and atail are

added to theends

RNA

transcriptwith capand tail

mRNA

TranscriptionAddition of cap and tail

Introns removed

Exons spliced together

Coding sequence

NUCLEUS

CYTOPLASM

Tail

Cap

The genetic code• The genetic code is written in

h f h b f



the sequence of the 4 bases of DNA: A, T, C, and G.

• Three bases read in sequencespecify one of the 20 aminoacids found in proteinmolecules.

• A codon is the 3-base sequencefor an amino acid.

• The message in the DNA istranscribed into an RNAmolecule, and then translatedinto a polypeptide

The Genetic Code II

• There are 64 (4X4X4) possible



triplet codes, but only 20 aminoacids.

• As seen in the table, more than1 triplet may code for the sameamino acid. This is no problem,

as long as no triplet can code formore than one amino acid.

• Note that several codons can

also act as start (AUG) or stop(UAA) signals.

Where can the amino acids be?

• A second type of RNA ist f RNA h f ti i



transfer RNA, whose function isto attach to a specific amino

acid and bring that amino acidsto the site where polypeptidesare being constructed.

• This RNA strand is twisted andbonded into the shape seen on

the right.• One end of the moleculeattached to a specific aminoacid.

• The other end has an exposedsequence of 3-bases. These arecalled the anticodon.

• How many kinds of tRNA mustthere be?

We must know our base pairs!!



• If we say 20 types of tRNA we are wrong!

• There must be a different tRNA molecule for each of thepossibletriplets. This means 64 anticodons.

The anticodons of the tRNAs each have a complimentarycodon in the mRNA. For example the codon AUG wouldbe the compliment of the anticodon UAC.

The role of Ribosomes

• The third type of RNA isi l RNA ( RNA)



risosomal RNA (rRNA).

• Ribosomes are the ‘decoding’units of the cell.

• Each ribosome consists of twosubunits, and is an assemblageof rRNA and proteins.

• Ribosomes have binding sitesfor both tRNA and mRNAmolecules.

Reading the Message

• An mRNA molecule attaches toa ribosome



a ribosome.• As the ribosome moves along

the mRNA, 3-base codons areexposed one at a time.• A tRNA with an anticodon that

is complimentary to the codonof the mRNA temporarily bondswith the mRNA.

• The ribosome positions themolecules so that this bondingoccurs.

• As the ribosome continues its journey along the mRNAadditional tRNAs bring theira.a. to the site of peptidesynthesis.

Elongation of the chain• As new amino acids are brought

to the ribosome, the growing



to the ribosome, the growingpeptide chain is attached to thenew amino acid by a peptidebond.

• Elongation of the chaincontinues until a stop codon isencountered. At that point thepeptide chain is released from

the tRNA.• A single mRNA can be readrepeatedly to make many copiesof a polypeptide.

• Once a tRNA gives up its amino

acid it can return to thecytoplasm and attach to anotherof its specified amino acid.

A Summary of the flow of GeneticInformation in a Cell

• Information is stored in thetriplet codes (codons) of DNA

• rRNA and proteins formribosomes



triplet codes (codons) of DNAnucleotides.

• This information is transcribedinto 3 types of RNA.

• mRNA carries the informationto assemble a polypeptide.

• In the nucleus, introns areremoved and the remainingexons spliced together to make afunctional mRNA strand.

• tRNA molecules attach tospecific amino acids.

ribosomes.• mRNA attaches to a ribosome

and the message is decodedwhen the anticodon of a tRNA isbonded to a mRNA codon.

• Subsequent amino acids are

attached to the growing peptidechain until a stop codon is reachand the chain is terminated.

• A summary of these events can

be seen in the next slide.



Mutation: When the Code is Miscopied• A mutation occurs when the code

doesn’t copy correctly, and aprotein is formed that doesn’t



protein is formed that doesn tfunction.

• If a base is substituted ordeleted, the triplet(s) aredifferent and so is the proteinformed.

• Mutations can also involvedinversion or deletion of largersections of the message.

• Substances that triggermutations are called mutagensand can be physical or chemicalin nature.

Transfer RNA

Amino Acid

ProteinForming

The DNA gene is first copiedand edited into a transcriptmade of RNA, employingsimilar nucleic acid bases,except that DNA’s thymine is



RIBOSOME

Messenger RNA

Anti-Codon

except that DNA s thymine isreplaced by uracil.

This messenger RNA (mRNA)version of the gene is then read

by cellular machinery, threeletters at a time, while tinycellular butlers known astransfer RNA’s (tRNA) fetch thespecified amino acids to bestrung together.

There arehundreds of possible AminoAcids

but these 20Amino Acids



Amino Acidsare used by life

on earth.

Amino AcidName

Triplet Code orCodon

3-LetterNickna

me

Glycine GGT,GGC,GGA,GGG Gly

Alanine GCT,GCC,GCA,GCG Ala

Valine GTT,GTC,GTA,GTG Val

Leucine TTG,TTA,CTT,CTC,CTA,CTG Leu

Isoleucine ATT,ATC,ATA Ileu



Serine TCT,TCC,TCA,TCG,AGT,AGC Ser

Threonine ACT,ACC,ACA,ACG ThrCysteine TGT,TGC Cys

Methionine ATG Met

Glutamic Acid GAA,GAG Glu

Aspartic Acid GAT,GAC,AAT,AAC Asp

Lysine AAA,AAG Lys

Arginine CGT,CGC,CGA,CGG,AGA,AGG Arg

Asparagine AAT,AAC Asn

Glutamine GAA,GAG Gln

Phenylalanine TTT,TTC Phe

Tyrosine TAT, TAC Tyr

Tryptophan TGG Trp

Proline CCT,CCC,CCA,CCG Pro

Terminator TAA,TAG,TGA End

20 types of

Aminoacyl-tRNA Synthetasesmatch 20 different amino acids totRNA’s with the correctanti-codon.

tRNA’s

RibosomesRibosomes



PSite

ASite

Largesubunit

Small subunit

mRNAmRNA

A U G C U A C U U C G

TranslationTranslation -- making proteinsmaking proteinsNuclear

b



membrane

TranscriptionTranscription

RNA ProcessingRNA Processing

TranslationTranslation

DNA

Pre-mRNA

mRNA

Ribosome

Protein

EukaryoticEukaryoticCellCell

TranslationTranslation• Three parts:



• Three parts:

1. initiationinitiation : start codon (AUG)2. elongationelongation :

3. terminationtermination : stop codon (UAG)

• Let’s make a PROTEIN!!!!PROTEIN!!!! .

TranslationTranslation



PSite

ASite

Largesubunit

Small subunit

mRNAmRNA


InitiationInitiationaa2

aa1



mRNAmRNA


2-tRNA

G A U

A

1-tRNA

U A Canticodon

hydrogenbonds codon

aa1 aa2

peptide bond

aa3

ElongationElongation



mRNAmRNA


1-tRNA 2-tRNA

U A C G A UA

anticodon

hydrogenbonds codon

3-tRNA

G A A

aa1

aa2

peptide bond

aa3



mRNAmRNAA U G C U A C U U C G

1-tRNA

2-tRNA

U A C

G A U

A

3-tRNA

G A A

Ribosomes move over one codon

(leaves)

aa1

aa2

peptide bonds

aa3

aa4




2-tRNA

G A U

A

3-tRNA

G A A

4-tRNA

G C U

A C U

aa1aa2

peptide bonds

aa3

aa4




2-tRNA

G A U

A

3-tRNA

G A A

4-tRNA

G C U

A C U

(leaves)


aa1aa2

peptide bonds

aa3

aa4

aa5



mRNAmRNAG C U A C U U C G A

3-tRNA

G A A

4-tRNA

G C U

A C U

U G A

5-tRNA

aa1aa2

peptide bonds

aa3 aa4

aa5



mRNAmRNAG C U A C U U C G A

3-tRNA

G A A 4-tRNA

G C U

A C U

U G A

5-tRNA


primaryprimarystructurestructureof a proteinof a protein

aa3

aa4aa5

aa200

aa199 TerminationTermination



mRNAmRNAA C A U G U

aa1

aa2

U

of a proteinof a protein

200-tRNA

U A GC U

terminatorterminatoror stopor stopcodoncodon

End ProductEnd Product• The end products of protein synthesis is aprimary structure of a proteinprimary structure of a protein



primary structure of a proteinprimary structure of a protein.

• A sequence of amino acidamino acidbondedtogether by peptide bondspeptide bonds.

aa1

aa2 aa3 aa4aa5

aa200

aa199

Question:Question:•• TheThe anticodonanticodonUACUACbelongs to abelongs to atRNAtRNAthatthat



gg

recognizes and binds to a particularrecognizes and binds to a particularaminoaminoacidacid..

•• What would be theWhat would be theDNA base codeDNA base codefor thisfor thisamino acid?amino acid?

Answer:Answer:•• tRNAtRNA -- UAC (UAC (anticodonanticodon))



tRNAtRNA UAC (UAC (anticodonanticodon))•• mRNAmRNA -- AUG (AUG (codoncodon))•• DNADNA -- TACTAC

Genetic Engineering l3

Documents

Transcript of Genetic Engineering l3