You are on page 1of 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 manipulation that involves molecular biological techniques. •Molecular Biology stands on the principle of Central Dogma :
Replication, Transcription Translation

DNA
Reverse Transcriptase

RNA

PROTEIN

•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 prokaryotes Attribute Organisms Cell wall Chromosome segregation meiosis Ribosome size Cell organelle Nuclear membrane Endoplasmic reticulum Golgi apparatus Mitochondria Chloroplast + + + + + Absent Eukaryotes Plants. Yes (plants) Mitotic spindle + 80 s Prokaryotes bacteria and cyanobacteria yes Cell membrane _ 70 s . animals and fungi No (animals).

Molecular biology: definition • Molecular biology is the study of molecular underpinnings of the process of replication. . transcription and translation of the genetic material.

Components involve in molecular biology DNA RNA Protein .

• Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring. • It is normally a stretch of DNA that codes for a type of protein or for an RNA chain that has a function in the organism.Gene : Unit of heredity • The DNA segments that carries genetic information are called genes. .

. such as proteins and RNA molecules.Deoxyribonucleic acid (DNA) • DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. • DNA is a set of blueprints needed to construct other components of cells.

Fig : DNA double helix . sugars and phosphate groups. with backbones made of base. • Chemically. DNA consists of two long polymers of simple units called nucleotides. • two strands run in opposite directions to each other and are therefore anti-parallel.• Two long strands makes the shape of a double helix.

• One major difference between DNA and RNA is the sugar. Ribose . with the 2deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.• The DNA double helix is stabilized by hydrogen bonds between the bases attached to the two strands.

and one nucleotide unit is 3.3 Å (0.Size: • The DNA chain is 22 to 26 Ångströms wide (2.33 nm) long.2 to 2. .6 nanometres).

translation . Transcription. DNA replication 3.1. DNA. RNA structure 2.

or T) Thymine (T) Sugar (deoxyribose) DNA nucleotide Polynucleotide Sugar-phosphate backbone Figure 10. C. made of long chains of nucleotides Phosphate group Nitrogenous base Sugar Phosphate group Nucleotide Nitrogenous base (A.DNA and RNA are polymers of nucleotides • DNA is a nucleic acid. G.2A .

C.2B .• DNA has four kinds of bases. and G Thymine (T) Cytosine (C) Adenine (A) Guanine (G) Pyrimidines Purines Figure 10. A. T.

DNA is a double-stranded helix • James Watson and Francis Crick worked out the three-dimensional structure of DNA. based on work by Rosalind Franklin .

3D .• Hydrogen bonds between bases hold the strands together: A and T. C and G Hydrogen bond Ribbon model Partial chemical structure Computer model Figure 10.

Review: The flow of genetic information in the cell is DNA—RNA---protein • The sequence of codons in DNA spells out the primary structure of a polypeptide – Polypeptides form proteins that cells and organisms use .

Mutations can change the meaning of genes • Mutations are changes in the DNA base sequence – caused by errors in DNA replication or by mutagens – change of a single DNA nucleotide causes sickle-cell disease .

Normal hemoglobin DNA Mutant hemoglobin DNA mRNA mRNA Normal hemoglobin Glu Sickle-cell hemoglobin Val .

• Types of mutations NORMAL GENE mRNA Protein Met Lys Phe Gly Ala BASE SUBSTITUTION Met Lys Phe Ser Ala BASE DELETION Missing Met Lys Leu Ala His .

•Chromosomal changes can be large or small Deletion Homologous chromosomes Duplication Inversion Reciprocal translocation Nonhomologous chromosomes .

DNA. protein overview . RNA.

RNA. 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 .under what conditions? . protein overview Questions about the genome in an organism: How much DNA.DNA.

Linking nucleotides 3’ 5’ 3’ Hydrogen bonds 3’ Linking nucleotides: N-H------N 3’ 3’ N-H------O The 3’-OH of one nucleotide is linked to the 5’-phosphate of the next? Whatnext nucleotide 3’ Thymine 3’ 2nm 3’ Adenine 3’ Cytosine 3’ 5’ Guanine 3’ .

protein structure 3’ 3’ C G 5’ .Base pairing 5’ 3’ A T 3’ Base pairing (Watson-Crick): 3’ C G A T 3’ A/T (2 hydrogen bonds) 3’ G/C (3 hydrogen bonds) Always pairing a purine and a pyrimidine yields a constant width 3’ DNA base composition: A + G = T + C (Chargaff’s rule) 3’ T A 3’ DNA RNA Mutations Amino acids.

DNA conventions 1. The 5’ end is to the left by convention 5’ -ATCGCAATCAGCTAGGTT-3’ 3’ -TAGCGTTAGTCGATCCAA-5’ sense (forward) antisense (reverse) 5’ -ATCGCAATCAGCTAGGTT. DNA is a right-handed helix 2.3’ 3’ -TAGCGTTAGTCGATCCAA.5’ .

2 x 109 bp.>50.20% > 50% 99% ? > 99% very .000 . Forces stabilizing DNA structure: Watson-Crick-H-bonding and base stacking (planar aromatic bases overlap geometrically and electronically → energy gain) 2. Some DNA molecules (plasmids) are circular and have no free ends: mtDNA bacterial DNA (only one circular chromosome) 4. Percentage of non-coding DNA varies greatly among organisms Organism small virus ‘typical’ virus little bacterium yeast human amphibians plants # Base pairs 4 x 103 3x 5 x 106 1 x 107 3. ~ 1 m contour length 3.000? ? 23.2 x 109 < 80 x 109 < 900 x 109 105 3000 6000 30. Average gene of 1000 bp can code for average protein of about 330 amino acids 5.7 x 106 bp.000 # Genes 3 Non-coding DNA very little 200 10 . Genomic DNAs are large molecules: Eschericia coli: 4. ~ 1 mm contour length Human: 3.DNA Structure Some more facts: 1.

Reading frames Reading frame (also open reading frame): The stretch of triplet sequence of DNA that potentially encodes a protein.each strand of RNA/DNA has three possible starting points (position one.a reading frame is not always easily recognizable . The reading frame is designated by the initiation or start codon and is terminated by a stop codon. or three): Position 1 CAG AUG AGG UCA GGC AUA gln met arg ser gly ile C AGA UGA GGU CAG GCA UA arg trp gly gln ala CA GAU GAG GUC AGG CAU A asp glu val arg his Position 2 Position 3 . two.mutations within an open reading frame that delete or add nucleotides can disrupt the reading frame (frameshift mutation): Wildtype CAG AUG AGG UCA GGC AUA GAG gln met arg Mutant ser gly ile glu Up to 30% of mutations causing humane disease are due to premature termination of translation (nonsense mutations or frameshift) CAG AUG AGU CAG GCA UAG AG gln met ser gln ala . .

Mutations Mutation: any heritable change in DNA Sources of mutation: Spontaneous mutations: mutations occur for unknown reasons Induced mutations: exposure to substance (mutagen) known to cause mutations. T ↔ G C ↔ A. free radicals Mutations may influence one or several base pairs a) Nucleotide substitutions (point mutation) 1) Transitions (Pu ↔ Pu. UV light.g. C ↔ G one to many bases can be involved frequently associated with repeated sequences (“hot spots”) lead to frameshift in protein-coding genes. e. A ↔ G 4 transversions: T ↔ A. X-rays. 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) . Py ↔ Py) 2) Transversions (Pu ↔ Py) b) Insertion or deletion (“indels”) - In-class exercise How many transition and transversion events are possible? 2 transitions: T ↔ C.

sickle cell anemia) the rate of nucleotide substitutions in humans is on the order of 1 per 100.g.sense: involves a change from a termination codon to one that codes for an amino acid .000. sickle cell anemia: mutation in ß-globin) c) Sense/nonsense substitution .000.Mutations Mutations 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 .neutral: protein still functions normally .missense: protein loses some functions (e.000 range varies from 1 in 10.causes amino acid change .000.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.000 every human has about 30 new mutations involving nucleotide substitutions mutation rate is about twice as high in male as in female meiosis .000 to 1 in 10.

Mutations A single amino acid substitution in a protein causes sickle-cell disease .

• It is encoded either in DNA or.What is Genome ? • Genome is the entirety of an organism's hereditary information. . in RNA. • The genome includes both the genes and the noncoding sequences of the DNA. for many types of virus.

000 19.comparative genome sizes of organisms organism Homo sapiens (human) Mus musculus (mouse) Drosophila melanogaster (fruit fly) Arabidopsis thaliana (plant) Caenorhabditis elegans (roundworm) Saccharomyces cerevisiae (yeast) Escherichia coli (bacteria) H.000 bases 1 gene /100.2 billion 2.1 million 4.000 ~25.6 billion 137 million 100 million 97 million 12.000 6000 3200 1700 average gene density 1 gene /100.000 bases 1 gene / 4000 bases 1 gene / 5000 bases 1 gene / 2000 bases 1 gene / 1400 bases 1 gene /1000 bases chromosome number 46 40 8 10 12 32 1 1 . influenzae (bacteria) Size (bp) 3.000 bases 1 gene / 9.6 million 1.000 25.000 13.8 million gene number ~25.

•To obtain the complete sequences of as many genomes as possible.Why Genome analysis ? •The prediction of genes in uncharacterised genomic sequences. •For Genetic modification. . •Genetic modification to develop new varieties at a faster rate like BT cotton and BT brinjal.

• • • . Each new DNA molecule will consist of one old stand. The gray strands in the figure to the right are new strands in the process of being assembled. the DNA in the nucleus of the cell must be duplicated. Since the DNA molecule consists of two complimentary stands. and a new complimentary strand. if those two strands separate and the right conditions are present. two new stands that are the compliments of the originals will be produced.Replication. Transcription. and Translation • Before a cell can divide.

Untwisting and replication of DNA • each strand is a template for a new strand helicase DNA polymerase .

. • This is seen in the figure below.Assembling the New Bases • The term semiconservative replication means that in the new DNA molecule there is one old and one new strand.

This allows parts of the DNA message to be replicated simultaneously in many locations.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. DNA polymerase adds new nucleotides . • • . while DNA ligase joints the DNA segments together. forming thousands of replication bubbles.

and it a bit more complicated than seen in the previous slide. . • This also insures that every new generation of individuals has the same genetic information as his/her parents.Why the fuss about DNA replication?? • The process of DNA replication involves a number of enzymes and proteins. so that each new cell in the body has the same set of genetic instructions as the cells that preceded it. • The important idea is that an exact duplication of the DNA message is required.

How can entire chromosomes be replicated during S phase? • DNA replication begins at many specific sites Parental strand Daughter strand Origin of replication Bubble Two daughter DNA molecules Figure 10.5A .

• Each strand of the double helix is oriented in the opposite direction 5′ end ′ 3′ end ′ P P P P P P P P 3′ end ′ 5′ end ′ Figure 10.5B .

• Cancer. aging DNA polymerase 5′ end ′ molecule Parental DNA 5′ ′ 3′ ′ 3′ ′ 5′ ′ Daughter strand synthesized continuously Daughter strand synthesized in pieces P 3′ ′ 5′ ′ 5′ ′ 3′ ′ P telomeres DNA ligase Overall direction of replication .• DNA polymerase works in only one direction • Telomere sequence s are lost with each replicatio n.

DNA replication • DNA replication. the basis for biological inheritance. • In the process of "replication" each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. . is a fundamental process occurring in all living organisms to copy their DNA. • Two identical DNA molecules have been produced from a single double-stranded DNA molecule.

• In a cell. • Unwinding of DNA at the origin. • In addition to DNA polymerase. DNA replication begins at specific locations in the genome. called "origins". forms a replication fork. the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand. . • Cellular proofreading that ensure near perfect fidelity for DNA replication. and synthesis of new strands. a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis.

The Replication of DNA THE CHEMISTRY OF DNA SYNTHESIS .

The Replication of DNA THE CHEMISTRY OF DNA SYNTHESIS DNA synthesis requires dNTPs and a primer:template junction .

DNA is synthesized by extending the 3’end of the primer Hydrolysis of pyrophosphate is the driving force for DNA Synthesis

G= -7 kcal/mole

DNA polymerases are processive enzymes; thus the rate of DNA synthesis is dramatically increased (~1000 bp/sec)

The thumb helps to maintain a strong association between the DNA polymerase and its substrate

Exonucleases proofread newly synthesized DNA

.

THE REPLICATION FORK Both strands of DNA are synthesized together at the replication fork .

DNA synthesis proceeds in a 5’ to 3’ direction and is semi-discontinuous. the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules. The structure of a DNA replication fork. called Okazaki fragments . Because both daughter DNA strands are polymerized in the 5'to-3' direction.

The initiation of a new strand of DNA requires an RNA primer .

the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template. The primase synthesizes a short polynucleotide in the 5'-to-3' direction and then stops.A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand RNA primer synthesis. making the 3' end of this primer available for the DNA polymerase . this enzyme can start a new polynucleotide chain by joining two or three nucleoside triphosphates together. Unlike DNA polymerase. A schematic view of the reaction catalyzed by DNA primase.

RNA primers must be removed to complete DNA replication .

DNA helicases unwind the double helix in advance of replication fork .

Topoisomerases remove supercoils produced by DNA unwinding at the replication fork .

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 REPLICATION Specific genomic DNA sequences direct the initiation of DNA Replication The replicon model of replication initiation .

Replicator sequences (origin of replication) include initiator binding sites and easily unwound DNA .

Eukaryotic chromosomes are replicated exactly once per cell cycle Incomplete replication causes chromosome breakage .

FINISHING REPLICATION Type II topoisomerases are required to separate daughter DNA molecules .

Lagging-strand synthesis is unable to copy the extreme ends of linear chromosomes .

One solution of the end problem is to use protein priming .

.

Only the part of the telomerase protein homologous to reverse transcriptase is shown here (green). G-rich telomere DNA sequence. . telomerase is unique in carrying its own RNA template with it at all times. A reverse transcriptase is a special form of polymerase enzyme that uses an RNA template to make a DNA strand. The telomerase is a protein– RNA complex that carries an RNA template for synthesizing a repeating.Telomerase Replicates the Ends of Eukaryotic Chromosomes The structure of telomerase.

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

Telomeres form a looped structure in the cell .

RNA Ribonucleic Acid .

usually Nitrogenous base (A. G. C. or U) Phosphate group Uracil (U) Sugar (ribose) .• RNA is also a nucleic acid – different sugar – U instead of T – Single strand.

Guanine . Cytosine.Structure of RNA Single stranded Ribose Sugar 5 carbon sugar Phosphate group Adenine. Uracil.

Types of RNA
Three main types Messenger RNA (mRNA) – transfers DNA code to ribosomes for translation. Transfer RNA (tRNA) – brings amino acids to ribosomes for protein synthesis. Ribosomal RNA (rRNA) – Ribosomes are made of rRNA and protein.

Table 14.2 Types of RNA Type of RNA Messenger RNA (mRNA) Functions in Nucleus, migrates to ribosomes in cytoplasm Function Carries DNA sequence information to ribosomes Provides linkage between mRNA and amino acids; transfers amino acids to ribosomes

Transfer RNA (tRNA)

Cytoplasm

Ribosomal RNA (rRNA)

Cytoplasm

Structural component of ribosomes

Ribonucleic acid (RNA)
• RNA is a biologically important type of molecule that consists of a long chain of nucleotide units. • Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate.

Double-stranded RNA • Double-stranded RNA (dsRNA) is RNA with two complementary strands. similar to the DNA found in all cells. . • dsRNA forms the genetic material of some viruses (double-stranded RNA viruses).

Types of RNA Type Messenger RNA Ribosomal RNA Transfer RNA Abbr mRNA rRNA tRNA Function Codes for protein Translation Translation Distribution All organisms All organisms All organisms in post-transcriptional modification Small nuclear RNA Y RNA Telomerase RNA snRNA Splicing and other Eukaryotes and functions archaea RNA processing. DNA Animals replication Telomere synthesis Most eukaryotes Regulatory RNAs Antisense RNA aRNA Transcriptional attenuation / mRNA degradation / mRNA All organisms stabilisation / Translation block .

This removes its introns—non-coding sections of the premRNA. the protein synthesis factories in the cell.Messenger RNA • mRNA carries information about a protein sequence to the ribosomes. • In eukaryotic cells. it is processed to mature mRNA. • It is coded so that every three nucleotides (a codon) correspond to one amino acid. . once precursor mRNA (pre-mRNA) has been transcribed from DNA.

which do not have nucleus and cytoplasm compartments.• The mRNA is then exported from the nucleus to the cytoplasm. . • In prokaryotic cells. mRNA can bind to ribosomes while it is being transcribed from DNA. where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA.

• .Transfer RNA • Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition • that site binds to a specific sequence on the messenger RNA chain through hydrogen bonding.

• rRNA molecules are synthesized in the nucleolus. 28S and 5S rRNA.8S. • The ribosome binds mRNA and carries out protein synthesis. • Eukaryotic ribosomes contain four different rRNA molecules: 18S. . ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. Several ribosomes may be attached to a single mRNA at any time. • In the cytoplasm. 5.Ribosomal RNA • Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. • rRNA is extremely abundant and makes up 80% of the 10 mg/ml RNA found in a typical eukaryotic cytoplasm.

Difference between RNA & DNA RNA RNA nucleotides contain ribose sugar RNA has the base uracil DNA DNA contains deoxyribose DNA has the base thymine presence of a hydroxyl group Lacks of a hydroxyl group at at the 2' position of the ribose the 2' position of the ribose sugar. RNA is usually singlestranded DNA is usually doublestranded . sugar.

The strand aaacguucgccc would be separated as aaa-cgu-ucg-ccc the amino acids would then be Lysine – Arginine – Serine .Proline .The Genetic Code This is the language of mRNA. Based on the 4 bases of mRNA. “Words” are 3 RNA sequences called codons.

Genetic Codes .

Virtually all organisms share the same genetic code Second Base “unity of life” U UUU UUC UUA UUG CUU CUC CUA CUG phe leu UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG C UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG A tyr stop stop his gln asn lys asp glu UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG G cys stop trp arg U C A G U C A G U C A G U C A G U ser First Base C leu pro Third B A AUU AUC ile AUA AUGmet (start) GUU GUC GUA GUG val ser arg thr G ala gly .

Phenylalanine UUU UUC Leucine UUA UUG Serine UCU UCC UCA UCG Tyrosine UAU UAC Stop UAA UAG UGA Arginine CGU CGC CGA CGG Lysine AAA AAG Cysteine UGU UGC Stop UGA Tryptophan UGG Leucine CUU CUC CUA CUG Isoleucine AUU AUC AUA Valine GUU GUC GUA GUG Proline CCU CCC CCA CCG Histidine CAU CAC Glutamine CAA CAG Methionine AUG Threonine ACU ACC ACA ACG Asparagine AAU AAC Serine AGU AGC Arginine AGG AGA Alanine GCU GCC GCA GCG Aspartic Acid GAU GAC Glutamic Acid GAA GAG Glycine GGU GGC GGA GGG .

RNA and Protein Synthesis
A. B. C. D. E. F. G. H. The Structure of RNA Types of RNA Transcription RNA Editing The Genetic Code Translation The Roles of RNA and DNA Genes and Proteins

Concept Map
RNA can be

Messenger RNA

Ribosomal RNA

Transfer RNA

also called

which functions to

also called

which functions to

also called

which functions to Bring amino acids to ribosome

mRNA

Carry instructions

rRNA

Combine with proteins

tRNA

from

to

to make up

DNA

Ribosome

Ribosomes

RNA structure
RNA ribonucleic acid 3 major types of RNA
messenger RNA (mRNA); template for protein synthesis transfer RNA (tRNA); adaptor molecules that decode the genetic code ribosomal RNA (rRNA); catalyzing the synthesis of proteins

4 bases A = Adenine U = Uracil C = Cytosine G = Guanine

Pyrimidine (C4N2H4)

Purine (C5N4H4)

Thymine (DNA) Nucleoside base + sugar (ribose)

Uracil (RNA) Nucleotide base + sugar
OO- PO4 O P

+ phosphate

--

OH
5’ CH2 4’

O
1’

O H

H

H
3’ OH

H
2’ OH

sugar

many self-complementary regions → RNA commonly exhibits an intricate secondary structure (relatively short.the folded RNA molecule is stabilized by interactions (e. hydrogen bonds and base stacking) .usually single stranded . double helical segments alternated with single stranded regions) .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: .complex tertiary interactions fold the RNA in its final three dimensional form .g.

other strand has contiguous base pairing F) junction three or more duplexes separated by single stranded regions G) pseudoknot tertiary interaction between bases of hairpin loop and outside bases .RNA structure Primary structure A) single stranded regions formed by unpaired nucleotides Secondary structure C B) duplex double helical RNA (A-form with 11 bp per turn) C) hairpin duplex bridged by a loop of unpaired nucleotides D) internal loop D E F B A G nucleotides not forming Watson-Crick base pairs E) bulge loop unpaired nucleotides in one strand.

RNA structure Primary structure Secondary structure C Tertiary structure D E F B A G .

RNA molecules comply to the laws of thermodynamics. possible interactions of RNA with solvent. no difficult experiments.RNA structure How to predict RNA secondary/tertiary structure? Probing RNA structure experimentally: . electron microscopy) .mutational analysis (introduction of specific mutations to test change in some function or protein-RNA interaction) Thermodynamic prediction of RNA structure: . not affected by interactions of the RNA and other molecules . ions. extremely variable regions cause problems with alignment . relatively easy to use. therefore it should be possible to deduce RNA structure from its sequence by finding the conformation with the lowest free energy . but not always accurate.chemical and enzymatic methods .Cons: large number of sequences to study preferred. this structure can be deduced by comparing the structures possible from their sequences . and proteins Comparative determination of RNA structure: . only sequences required. structure constrains in fully conserved regions cannot be inferred.basic assumption: secondary structure of a functional RNA will be conserved in the evolution of the molecule (at least more conserved than the primary structure).Pros: very powerful in finding secondary structure. does not rely on alignments .Pros: only one sequence required. when a set of homologous sequences has a certain structure in common.physical methods (single crystal X-ray diffraction.Cons: thermodynamic data experimentally determined.

RNA AND PROTEIN SYNTHESIS .

. • Protein synthesis involves two processes: transcription and translation.DNA carries information that can be used to construct the proteins which form structures and regulate the body’s activities. • In translation the RNA message is used to assemble amino acids into a protein chain. • In transcription the DNA message is converted into an RNA molecule.

RNA processing 3.Protein ---RNA . Translation • DNA ---RNA --. Transcription 2. • 3 phases phases: 1.How does our cell makes very important proteins • The production (synthesis) of proteins proteins.

Our cell must first make RNA • Question: • How does RNA (ribonucleic acid) differ from DNA (deoxyribonucleic acid) acid)? .Before making proteins.

RNA differs from DNA 1. RNA has a sugar ribose DNA has a sugar deoxyribose 2. RNA molecule is single-stranded singleDNA is double-stranded double- . RNA contains uracil (U) DNA has thymine (T) 3.

.

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 to the cafeteria lady. .

Messenger RNA (mRNA) mRNA start codon A U G G G C U C C A U C G G C G C A U A A codon 1 protein methionine codon 2 glycine codon 3 serine codon 4 isoleucine codon 5 glycine codon 6 alanine codon 7 stop codon Primary structure of a protein aa1 aa2 aa3 peptide bonds aa4 aa5 aa6 .A.

If the cell is a school… • The Nucleus is the school office • The Nucleolus is the principal’s office • The DNA is the principal • Ribosomes are the cafeteria ladies • mRNA is the email from the principal to the cafeteria lady .

Gets the right parts to make the right protein according to mRNA instructions .rRNA • Part of the structure of a ribosome • Helps in protein production tRNA A go-getter.

• Introns bad…… Exons good! . • End product is a mature RNA molecule that leaves the nucleus to the cytoplasm.RNA Processing • Introns are pulled out and exons come together.

RNA Processing pre-RNA molecule exon intron exon intron exon intron exon splicesome intron exon splicesome exon exon exon exon Mature RNA molecule .

• This process occurs before the message leaves the nucleus. the message contains unwanted base sequences. . • The ‘junk’ sequences (called introns) are removed from the message and the remaining sequences (exons) are linked together to produce a sequence of codons that will translate into a polypeptide.Cleaning up the Message • When the genetic message is copied to make mRNA.

RNA polymerase binds to DNA and separates the DNA strands. During transcription. RNA polymerase then uses one strand of DNA as a template from which nucleotides are assembled into a strand of mRNA. . a process called transcription.Transcription RNA molecules are produced by copying part of the nucleotide sequence of DNA into complementary sequence in RNA.

where it binds and begins transcription. Some parts are removed (introns) .mRNA How Does it Work? RNA Polymerase looks for a region on the DNA known as a promoter. RNA strands are then edited. .which are not expressed – and other that are left are called exons or expressed genes.

which is translated into the polypeptide DNA TRANSCRIPTION RNA TRANSLATION Protein .• The information constituting an organism’s genotype is carried in its sequence of bases – The DNA is transcribed into RNA.

Prokaryotic gene expression .

RNA polymerase binds to the -10 and 35 regions of the promoter relative to the start site of transcription (+1) promoter operator .In prokaryotes.

.

Reverse transcription • Reverse transcribing viruses replicate their genomes by reverse 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.

.

• DNA transcription RNA. is the process of creating an equivalent RNA copy of a sequence of DNA. • Transcription is the first step leading to gene expression. a DNA sequence is read by RNA polymerase. which produces a complementary. . reverse transcription • During transcription. antiparallel RNA strand.Transcription • Transcription. • Transcription results in an RNA complement that includes uracil (U) instead of thymine (T).

Transcription process • The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. the transcribed gene may encode for either rRNA or tRNA. or other ribozymes. • Alternatively. • This mRNA will be used to create that protein via the process of translation. the result of transcription is messenger RNA (mRNA). • If the gene transcribed encodes for a protein. other components of the protein-assembly process. • A DNA transcription unit encoding for protein (the coding sequence) and regulatory sequences that direct and regulate the synthesis of that protein. .

• The other DNA strand is called the coding strand.• DNA is read from 3' → 5' during transcription. . • only one of the two DNA strands. • the complementary RNA is created from the 5' → 3' direction. is used for transcription because RNA is only single-stranded. called the template strand.

Transcription produces genetic messages in the form of mRNA RNA polymerase RNA nucleotide Direction of transcription Template strand of DNA Newly made RNA .

RNA polymerase

• In transcription, DNA helix unzips
– RNA nucleotides line up along one strand of DNA, following the basepairing rules – single-stranded messenger RNA peels away and DNA strands rejoin
Figure 10.9B

DNA of gene

Promoter DNA Initiation

Terminator DNA

Elongation

Area shown in Figure 10.9A

Termination Growing RNA

Completed RNA RNA polymerase

RNA transcripts of DNA

Eukaryotic RNA is processed before leaving the nucleus
• Noncoding segments, introns, are spliced out • A cap and a tail are added to the ends
Exon Intron DNA Cap RNA transcript with cap and tail Transcription Addition of cap and tail Exon Intron Exon

Introns removed

Tail

Exons spliced together mRNA Coding sequence NUCLEUS

CYTOPLASM

C. • The message in the DNA is transcribed into an RNA molecule. and then translated into a polypeptide . • A codon is the 3-base sequence for an amino acid. • Three bases read in sequence specify one of the 20 amino acids found in protein molecules. and G. T.The genetic code • The genetic code is written in the sequence of the 4 bases of DNA: A.

• As seen in the table. .The Genetic Code II • There are 64 (4X4X4) possible triplet codes. • Note that several codons can also act as start (AUG) or stop (UAA) signals. as long as no triplet can code for more than one amino acid. This is no problem. more than 1 triplet may code for the same amino acid. but only 20 amino acids.

whose function is to attach to a specific amino acid and bring that amino acids to the site where polypeptides are being constructed. • How many kinds of tRNA must there be? . • The other end has an exposed sequence of 3-bases. • One end of the molecule attached to a specific amino acid.Where can the amino acids be? • A second type of RNA is transfer RNA. These are called the anticodon. • This RNA strand is twisted and bonded into the shape seen on the right.

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 the possible triplets. This means 64 anticodons. The anticodons of the tRNAs each have a complimentary codon in the mRNA. . For example the codon AUG would be the compliment of the anticodon UAC.

. • Ribosomes have binding sites for both tRNA and mRNA molecules.The role of Ribosomes • The third type of RNA is risosomal RNA (rRNA). • Each ribosome consists of two subunits. and is an assemblage of rRNA and proteins. • Ribosomes are the ‘decoding’ units of the cell.

• As the ribosome continues its journey along the mRNA additional tRNAs bring their a. • A tRNA with an anticodon that is complimentary to the codon of the mRNA temporarily bonds with the mRNA. • As the ribosome moves along the mRNA. .a. to the site of peptide synthesis.Reading the Message • An mRNA molecule attaches to a ribosome. • The ribosome positions the molecules so that this bonding occurs. 3-base codons are exposed one at a time.

• A single mRNA can be read repeatedly to make many copies of a polypeptide. • Once a tRNA gives up its amino acid it can return to the cytoplasm and attach to another of its specified amino acid. • Elongation of the chain continues until a stop codon is encountered. the growing peptide chain is attached to the new amino acid by a peptide bond.Elongation of the chain • As new amino acids are brought to the ribosome. . At that point the peptide chain is released from the tRNA.

A Summary of the flow of Genetic Information in a Cell • Information is stored in the triplet codes (codons) of DNA nucleotides. • Subsequent amino acids are attached to the growing peptide chain until a stop codon is reach and the chain is terminated. • tRNA molecules attach to specific amino acids. introns are removed and the remaining exons spliced together to make a functional mRNA strand. • In the nucleus. • This information is transcribed into 3 types of RNA. . • rRNA and proteins form ribosomes. • mRNA attaches to a ribosome and the message is decoded when the anticodon of a tRNA is bonded to a mRNA codon. • A summary of these events can be seen in the next slide. • mRNA carries the information to assemble a polypeptide.

.

• Substances that trigger mutations are called mutagens and can be physical or chemical in nature. the triplet(s) are different and so is the protein formed. . and a protein is formed that doesn’t function.Mutation: When the Code is Miscopied • A mutation occurs when the code doesn’t copy correctly. • If a base is substituted or deleted. • Mutations can also involved inversion or deletion of larger sections of the message.

employing similar nucleic acid bases.Amino Acid Transfer RNA Protein Forming The DNA gene is first copied and edited into a transcript made of RNA. except that DNA’s thymine is replaced by uracil. This messenger RNA (mRNA) version of the gene is then read by cellular machinery. while tiny cellular butlers known as transfer RNA’s (tRNA) fetch the specified amino acids to be strung together. three letters at a time. Anti-Codon Messenger RNA RIBOSOME .

.There are hundreds of possible Amino Acids but these 20 Amino Acids are used by life on earth.

AGG AAT.AGC ACT. AGA.CTT.AAC AAA.CCG TAA.TGC ATG GAA.ACC.GTA.AAG CGT.C TA.GAG TTT. tRNA’s .A GT.TCG.GAC.CTG ATT.TTA.GAG GAT.ATC.ACG TGT.GGG GCT.Amino Acid Name Glycine Alanine Valine Leucine Isoleucine Serine Threonine Cysteine Methionine Glutamic Acid Aspartic Acid Lysine Arginine Asparagine Glutamine Phenylalanine Tyrosine Tryptophan Proline Terminator Triplet Code or Codon GGT.ACA.CCA.GCG GTT.GGA.GTG TTG.TCA.CGG.CGC.GCA.AAC GAA.TTC TAT.AAT.CCC.GGC.TCC.ATA TCT. TAC TGG CCT.GCC.CGA.CTC.TGA 3-Letter Nickna me Gly Ala Val Leu Ileu Ser Thr Cys Met Glu Asp Lys Arg Asn Gln Phe Tyr Trp Pro End 20 types of Aminoacyl-tRNA Synthetases match 20 different amino acids to tRNA’s with the correct anti-codon.GTC.TAG.

Ribosomes Large subunit P Site A Site mRNA A U G Small subunit C U A C U U C G .

making proteins DNA Transcription Nuclear membrane Pre-mRNA Eukaryotic Cell RNA Processing mRNA Ribosome Translation Protein .Translation .

.Translation • Three parts: 1. termination stop codon (UAG) termination: • Let’s make a PROTEIN!!!! PROTEIN!!!!. initiation start codon (AUG) initiation: 2. elongation elongation: 3.

Translation Large subunit P Site A Site mRNA A U G Small subunit C U A C U U C G .

Initiation aa1 aa2 2-tRNA 1-tRNA anticodon hydrogen bonds U A C A U G codon G A U C U A C U U C G A mRNA .

Elongation aa1 peptide bond aa3 aa2 3-tRNA 1-tRNA anticodon 2-tRNA G A A hydrogen bonds U A C A U G codon G A U C U A C U U C G A mRNA .

aa1 peptide bond aa3 aa2 1-tRNA U A C (leaves) 2-tRNA 3-tRNA G A A A U G G A U C U A C U U C G A mRNA Ribosomes move over one codon .

aa1 peptide bonds aa4 aa2 aa3 4-tRNA 2-tRNA 3-tRNA G C U A U G G A U G A A C U A C U U C G A A C U mRNA .

aa1 peptide bonds aa2 aa3 aa4 2-tRNA 4-tRNA G A U (leaves) 3-tRNA G C U A U G G A A C U A C U U C G A A C U mRNA Ribosomes move over one codon .

aa1 peptide bonds aa2 aa3 aa4 aa5 5-tRNA U G A 3-tRNA 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA .

aa1 aa2 peptide bonds aa3 aa5 aa4 5-tRNA 3-tRNA U G A 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA Ribosomes move over one codon .

aa4 aa5 aa199 aa200 Termination aa3 primary structure aa2 of a protein aa1 200-tRNA terminator or stop codon A C U mRNA C A U G U U U A G .

aa2 aa1 aa3 aa4 aa5 aa199 aa200 .End Product • The end products of protein synthesis is a primary structure of a protein protein. • A sequence of amino acid bonded together by peptide bonds bonds.

acid.Question: • The anticodon UAC belongs to a tRNA that recognizes and binds to a particular amino acid. • What would be the DNA base code for this amino acid? .

Answer: • tRNA • mRNA • DNA .UAC (anticodon) (anticodon) .AUG (codon) (codon) .TAC .