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, animals and fungi No (animals); Yes (plants) Mitotic spindle + 80 s Prokaryotes bacteria and cyanobacteria yes Cell membrane _ 70 s

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

Gene : Unit of heredity

The DNA segments that carries genetic information are called genes. 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. Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring.

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, such as proteins and RNA molecules.

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

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

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

Ribose

Size:

The DNA chain is 22 to 26 ngstrms wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 (0.33 nm) long.

1. DNA, RNA structure 2. DNA replication 3. Transcription, translation

DNA and RNA are polymers of nucleotides

DNA is a nucleic acid, made of long chains of nucleotides
Phosphate group Nitrogenous base Sugar Phosphate group Nucleotide Nitrogenous base (A, G, C, or T)

Thymine (T)

Sugar (deoxyribose)

DNA nucleotide Polynucleotide Sugar-phosphate backbone Figure 10.2A

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

Thymine (T)

Cytosine (C)

Adenine (A)

Guanine (G)

Pyrimidines

Purines

Figure 10.2B

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

 Hydrogen bonds between bases hold the strands together: A and T, C and G
Hydrogen bond

Ribbon model

Partial chemical structure

Computer model
Figure 10.3D

Review: The flow of genetic information in the cell is DNARNA---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, 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 - under what conditions?

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

Thymine
3

2nm
3

Adenine
3

Cytosine
3

Guanine

Base pairing
5

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

DNA base composition: A + G = T + C (Chargaffs rule)

T A
3

DNA RNA Mutations Amino acids, protein structure

3
3

C G
5

DNA conventions

1. DNA is a right-handed helix 2. The 5 end is to the left by convention 5 -ATCGCAATCAGCTAGGTT-3 3 -TAGCGTTAGTCGATCCAA-5 sense (forward) antisense (reverse)

5 -ATCGCAATCAGCTAGGTT- 3 3 -TAGCGTTAGTCGATCCAA- 5

DNA Structure
Some 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 106 bp; ~ 1 mm contour length Human: 3.2 x 109 bp; ~ 1 m contour length 3. Some DNA molecules (plasmids) are circular and have no free ends: mtDNA bacterial 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 small virus typical virus little bacterium yeast human amphibians plants # Base pairs 4 x 103 3x 5 x 106 1 x 107 3.2 x 109 < 80 x 109 < 900 x 109 105 3000 6000 30,000? ? 23,000 - >50,000 # Genes 3 Non-coding DNA very little 200 10 - 20% > 50% 99% ? > 99% very

Reading frames
Reading frame (also open reading frame): The stretch of triplet sequence of DNA that potentially encodes a protein. The reading frame is designated by the initiation or start codon and is terminated by a stop codon. - a reading frame is not always easily recognizable - each strand of RNA/DNA has three possible starting points (position one, two, 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

- 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, 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) b) Insertion or deletion (indels) -

In-class exercise How many transition and transversion events are possible?

2 transitions: T C; A G 4 transversions: T A; T G C A; C G 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)

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 - causes amino acid change - neutral: protein still functions normally - missense: protein loses some functions (e.g. sickle cell anemia: mutation in -globin) c) Sense/nonsense substitution - sense: involves a change from a termination codon to 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

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

What is Genome ?

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

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. influenzae (bacteria)

Size (bp)
3.2 billion 2.6 billion 137 million 100 million 97 million 12.1 million 4.6 million 1.8 million

gene number
~25,000 ~25,000 13,000 25,000 19,000 6000 3200 1700

average gene density

1 gene /100,000 bases 1 gene /100,000 bases 1 gene / 9,000 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

Why Genome analysis ?

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

Replication, Transcription, and Translation

Before a cell can divide, the DNA in the nucleus of the cell must be duplicated. Since the DNA molecule consists of two complimentary stands, if those two strands separate and the right conditions are present, two new stands that are the compliments of the originals will be produced. Each new DNA molecule will consist of one old stand, and a new complimentary strand. The gray strands in the figure to the right are new strands in the process 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, forming thousands of replication bubbles. This allows parts of the DNA message to be replicated simultaneously in many locations. DNA polymerase adds new nucleotides , while DNA ligase joints the DNA segments 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 DNA message is required, so that each new cell in the body has the same set of genetic instructions as the cells that preceded it. This also insures that every new generation of individuals has the same genetic information as his/her parents.

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
3 end 5 end

Figure 10.5B

 DNA polymerase works in only one direction

Telomere sequence s are lost with each replicatio n. Cancer, aging

DNA polymerase 5 end molecule Parental DNA 5 3

3 5 Daughter strand synthesized continuously Daughter strand synthesized in pieces

3 5

5 3

telomeres

DNA ligase

Overall direction of replication

DNA replication
DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. In the process of "replication" each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Two identical DNA molecules have been produced from 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 a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. Cellular proofreading that ensure near perfect fidelity for DNA replication.

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 3end 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 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

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

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

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

Telomerase Replicates the Ends of Eukaryotic Chromosomes

The structure of telomerase.

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

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

Telomeres form a looped structure in the cell

RNA
Ribonucleic Acid

 RNA is also a nucleic acid

different sugar U instead of T Single strand, usually
Nitrogenous base (A, G, C, or U) Phosphate group

Uracil (U)

Sugar (ribose)

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

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

Messenger RNA
mRNA carries information about a protein sequence to the ribosomes, 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) has been transcribed from DNA, it is processed to mature mRNA. This removes its intronsnon-coding sections of the premRNA.

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

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.

Ribosomal RNA
Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. rRNA molecules are synthesized in the nucleolus. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time. 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. sugar. RNA is usually singlestranded DNA is usually doublestranded

The Genetic Code

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

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

ser

First Base

leu

pro

Third B

AUU AUC ile AUA AUGmet (start) GUU GUC GUA GUG val

ser arg

thr

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
3 OH

H
2 OH

sugar

Base interactions in RNA

Base pairing: U/A/(T) (2 hydrogen bonds) G/C (3 hydrogen bonds)

RNA base composition: A+G/ U+C = Chargaffs 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
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, 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

Secondary structure C

Tertiary structure

D E F B A G

RNA structure
How to predict RNA secondary/tertiary structure?
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 be possible to deduce RNA structure from its sequence by finding the conformation with the lowest free energy - Pros: only one sequence required; no difficult experiments; does not rely on alignments - 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 the evolution 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 can be deduced by comparing the structures possible from their sequences - Pros: very powerful in finding secondary structure, relatively easy to use, only sequences required, 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 AND PROTEIN SYNTHESIS

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

How does our cell makes very important proteins

The production (synthesis) of proteins proteins. 3 phases phases: 1. Transcription 2. RNA processing 3. Translation DNA ---RNA --- Protein ---RNA

Before making proteins, Our cell must first make RNA

Question: How does RNA (ribonucleic acid) differ from DNA (deoxyribonucleic acid) acid)?

RNA differs from DNA

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

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.

A. 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

If the cell is a school

The Nucleus is the school office The Nucleolus is the principals office The DNA is the principal Ribosomes are the cafeteria ladies mRNA is the email from the principal to the cafeteria lady

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

tRNA
A go-getter. Gets the right parts to make the right protein according to mRNA instructions

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

RNA Processing
pre-RNA molecule exon intron exon intron exon

intron
exon
splicesome

intron
exon
splicesome

exon

exon

exon

exon

Mature RNA molecule

Cleaning up the Message

When the genetic message is copied to make mRNA, 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. This process occurs before the message leaves the nucleus.

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

mRNA How Does it Work?

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

 The information constituting an organisms genotype is carried in its sequence of bases

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

TRANSCRIPTION

RNA

TRANSLATION
Protein

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 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.

Transcription
Transcription, is the process of creating an equivalent RNA copy of a sequence of DNA. Transcription is the first step leading to gene expression. DNA
transcription

RNA.

reverse transcription

During transcription, a DNA sequence is read by RNA polymerase, which produces a complementary, antiparallel RNA strand. 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. 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-assembly process, or other ribozymes. A DNA transcription unit encoding for protein (the coding sequence) and regulatory sequences that direct and regulate the synthesis of that protein.

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

The genetic code

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

The Genetic Code II

There are 64 (4X4X4) possible triplet codes, but only 20 amino acids. As seen in the table, more than 1 triplet may code for the same amino acid. This is no problem, as long as no triplet can code for more 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 is transfer RNA, whose function is to attach to a specific amino acid and bring that amino acids to the site where polypeptides are being constructed. This RNA strand is twisted and bonded into the shape seen on the right. One end of the molecule attached to a specific amino acid. The other end has an exposed sequence of 3-bases. These are called the anticodon. How many kinds of tRNA must there 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 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.

The role of Ribosomes

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

Reading the Message

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

Elongation of the chain

As new amino acids are brought to the ribosome, the growing peptide chain is attached to the new amino acid by a peptide bond. Elongation of the chain continues until a stop codon is encountered. At that point the peptide chain is released from the tRNA. 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.

A Summary of the flow of Genetic Information in a Cell

Information is stored in the triplet codes (codons) of DNA nucleotides. This information is transcribed into 3 types of RNA. mRNA carries the information to assemble a polypeptide. In the nucleus, introns are removed and the remaining exons spliced together to make a functional mRNA strand. tRNA molecules attach to specific amino acids. 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. Subsequent amino acids are attached to the growing peptide chain until a stop codon is reach and 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 doesnt copy correctly, and a protein is formed that doesnt function. If a base is substituted or deleted, the triplet(s) are different and so is the protein formed. Mutations can also involved inversion or deletion of larger sections of the message. Substances that trigger mutations are called mutagens and can be physical or chemical in nature.

Amino Acid Transfer RNA Protein Forming

The DNA gene is first copied and edited into a transcript made of RNA, employing similar nucleic acid bases, except that DNAs thymine is replaced by uracil. This messenger RNA (mRNA) version of the gene is then read by cellular machinery, three letters at a time, while tiny cellular butlers known as transfer RNAs (tRNA) fetch the specified amino acids to be strung together.

Anti-Codon

Messenger RNA

RIBOSOME

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

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,GGC,GGA,GGG GCT,GCC,GCA,GCG GTT,GTC,GTA,GTG TTG,TTA,CTT,CTC,C TA,CTG ATT,ATC,ATA TCT,TCC,TCA,TCG,A GT,AGC ACT,ACC,ACA,ACG TGT,TGC ATG GAA,GAG GAT,GAC,AAT,AAC AAA,AAG CGT,CGC,CGA,CGG, AGA,AGG AAT,AAC GAA,GAG TTT,TTC TAT, TAC TGG CCT,CCC,CCA,CCG TAA,TAG,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 tRNAs with the correct anti-codon. tRNAs

Ribosomes
Large subunit

P Site

A Site
mRNA

A U G
Small subunit

C U A C U U C G

Translation - making proteins

DNA
Transcription Nuclear membrane

Pre-mRNA

Eukaryotic Cell

RNA Processing

mRNA Ribosome
Translation

Protein

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

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

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.
aa2 aa1 aa3 aa4 aa5
aa199

aa200

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

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