documentig

DNA, RNA, Genetic DNA, RNA, Genetic code, and code, and

biosynthesis of biosynthesis of proteinsproteins

DNA vs. RNADNA vs. RNA1. Number of strands1. Number of strands

2. Nucleotide composition2. Nucleotide composition

3. Synthesis3. Synthesis

4. Location4. Location

5. Functions5. Functions

The GeneThe Gene

A gene is traditionally defined as a unit of A gene is traditionally defined as a unit of heredityheredity

It’s a DNA segment that encodes a single It’s a DNA segment that encodes a single polypeptide chain (1 gene-one-polypeptide polypeptide chain (1 gene-one-polypeptide hypothesishypothesis

Careful!Careful!: Alternative sequences encoding : Alternative sequences encoding same polypeptide/same gene can produse same polypeptide/same gene can produse different polypeptides. different polypeptides.

Gene operationGene operation

Regulatory regionRegulatory region Coding/structural regionCoding/structural region

How is genetic information passed How is genetic information passed from DNA to a protein?from DNA to a protein?

Key qnsKey qns How is genetic information passed on from How is genetic information passed on from

generation to generation, or just cell to cell? generation to generation, or just cell to cell? How can a “group of letters" determine what How can a “group of letters" determine what

proteins are made in the cell and direct the proteins are made in the cell and direct the cell's activities? cell's activities?

According to the central dogma of molecular According to the central dogma of molecular genetics, the function of DNA is to genetics, the function of DNA is to store information and pass it on to RNA, store information and pass it on to RNA,

while the function of RNA is while the function of RNA is to read, decode and use the information to read, decode and use the information

received from DNA to make proteins. received from DNA to make proteins.

Central Dogma of Molecular Biology

DNA mRNA

PermanentGeneticarchive

Same in all cells

transcription

translation

rev transcription

replication

TransientCarrierof specificinformation

Different in different cells

Transient expressionof informationfor action in cellDifferent in different cells

Protein

DNA ReplicationDNA Replication

Is the process by which a replica, or identical Is the process by which a replica, or identical copy, of DNA is made. copy, of DNA is made.

Replication occurs Replication occurs every time a cell dividesevery time a cell divides so that information can be preserved and so that information can be preserved and handed down to offspring. handed down to offspring.

mRNA transcriptionmRNA transcription

Is the process by which the genetic messages Is the process by which the genetic messages contained in DNA are "read" or transcribed. contained in DNA are "read" or transcribed.

The product of transcription, known as The product of transcription, known as messenger RNA (mRNA), leaves the cell messenger RNA (mRNA), leaves the cell nucleus and carries the message to the sites of nucleus and carries the message to the sites of protein synthesis. protein synthesis.

TranslationTranslation

Is the process by which the genetic messages Is the process by which the genetic messages carried by mRNA are decoded and used to carried by mRNA are decoded and used to build proteins. build proteins.

Challenge!Challenge!

For instance, if you eat a piece of beef and For instance, if you eat a piece of beef and the cells of your pancreas need to secrete a the cells of your pancreas need to secrete a digestive enzyme, then the one gene for that digestive enzyme, then the one gene for that enzyme will be transcribed from DNA to enzyme will be transcribed from DNA to mRNA and then to the digestive enzyme to mRNA and then to the digestive enzyme to digest the piece of beef.digest the piece of beef.

Do we need DNA to replicate for this?Do we need DNA to replicate for this?

NO!! NO!!

The only reason a cell has for replicating its DNA is The only reason a cell has for replicating its DNA is if it's going to divideif it's going to divide

Replication processReplication process

DNA replication begins with a partial unwinding of DNA replication begins with a partial unwinding of the double helix at an area known as the the double helix at an area known as the replication replication fork (fork (under DNA helicaseunder DNA helicase) .) .

As the two DNA strands separate ("unzip") and the As the two DNA strands separate ("unzip") and the bases are exposed, the enzyme bases are exposed, the enzyme DNA polymeraseDNA polymerase moves into position at the point where synthesis will moves into position at the point where synthesis will begin begin

The start point for DNA polymerase is a short The start point for DNA polymerase is a short segment of RNA known as an segment of RNA known as an RNA primerRNA primer..

The primer is "laid down" complementary to The primer is "laid down" complementary to the DNA template by an enzyme known as the DNA template by an enzyme known as RNA polymerase RNA polymerase or or PrimasePrimase

The DNA polymerase (once it has reached its The DNA polymerase (once it has reached its starting point as indicated by the primer) then starting point as indicated by the primer) then adds nucleotides one by one in an exactly adds nucleotides one by one in an exactly complementary manner, A to T and G to Ccomplementary manner, A to T and G to C


DNA polymerase is described as being "template DNA polymerase is described as being "template dependent" in that it will dependent" in that it will "read""read" the sequence of the sequence of bases on the template strand and then bases on the template strand and then "synthesize""synthesize" the complementary strand the complementary strand

DNA polymerase catalyzes the formation of the DNA polymerase catalyzes the formation of the hydrogen bonds between each arriving nucleotide and hydrogen bonds between each arriving nucleotide and the nucleotides on the template strand. the nucleotides on the template strand.

DNA polymerase also catalyzes the reaction between DNA polymerase also catalyzes the reaction between the 5' phosphate on an incoming nucleotide and the free the 5' phosphate on an incoming nucleotide and the free 3' OH on the growing polynucleotide 3' OH on the growing polynucleotide


The last step is for an enzyme to come along The last step is for an enzyme to come along and remove the existing RNA primers and and remove the existing RNA primers and then fill in the gaps with DNA. then fill in the gaps with DNA.


TranscriptionTranscription

Types of RNATypes of RNA1.1. Ribosomal RNAsRibosomal RNAs-- Exist outside the nucleus in the cytoplasm of a Exist outside the nucleus in the cytoplasm of a

cell in structures calledcell in structures called ribosomes ribosomes. . Ribosomes are small, granular structures where Ribosomes are small, granular structures where

protein synthesis takes place. protein synthesis takes place. Each ribosome is a complex consisting of about Each ribosome is a complex consisting of about

60% ribosomal RNA 60% ribosomal RNA (rRNA)(rRNA) and 40% protein and 40% protein

2. Transfer RNAs2. Transfer RNAs-- The function of transfer RNAs The function of transfer RNAs (tRNA)(tRNA) is to is to

deliver amino acids one by one to protein chains deliver amino acids one by one to protein chains growing at ribosomes.growing at ribosomes.

3. Messenger RNAs3. Messenger RNAs-- Are the nucleic acids that "record" information Are the nucleic acids that "record" information

from DNA in the cell nucleus and carry it to the from DNA in the cell nucleus and carry it to the ribosomes and are known as messenger RNAs ribosomes and are known as messenger RNAs (mRNA).(mRNA).

TranscriptionTranscription

mRNA is synthesized by the mRNA is synthesized by the transcription of a portion of one strand transcription of a portion of one strand of DNA (the active strand) to produce a of DNA (the active strand) to produce a complementary single RNA strand complementary single RNA strand

Synthesis of mRNA takes place in the Synthesis of mRNA takes place in the nucleus under the enzyme nucleus under the enzyme RNA RNA polymerase IIpolymerase II

RNA TranscriptionRNA Transcription((Nucleus)Nucleus)

Transcription is initiated by signals from Transcription is initiated by signals from cytoplasm. cytoplasm.

One strand of the DNA is activated (or One strand of the DNA is activated (or suppressing factor removed!) suppressing factor removed!) induction of local unwinding of the DNA induction of local unwinding of the DNA

helix helix RNA polymerase IIRNA polymerase II binds at DNA promoter binds at DNA promoter

region for transcription initiationregion for transcription initiation

Synthesis of mRNASynthesis of mRNA

Post-transcriptional mRNA processingPost-transcriptional mRNA processing Poly adenylation (3’ end)-up to 200 bases in Poly adenylation (3’ end)-up to 200 bases in

eukaryoteseukaryotes Methylation (5’ end)-a capMethylation (5’ end)-a cap Splicing-removal of introns Splicing-removal of introns

mRNA leaves nucleus and moves to mRNA leaves nucleus and moves to cytoplasmcytoplasm


Protein synthesisProtein synthesis

The information (message) in the mRNA is The information (message) in the mRNA is decoded into an amino acid sequence of a decoded into an amino acid sequence of a polypepetide polypepetide

Genetic codeGenetic code DNA transfers information to mRNA in the form DNA transfers information to mRNA in the form

of a code defined by a sequence of nucleotides of a code defined by a sequence of nucleotides bases. bases.

During protein synthesis, ribosomes move along During protein synthesis, ribosomes move along the mRNA molecule and "read" its sequence the mRNA molecule and "read" its sequence (three nucleotides at a time) called codons, from (three nucleotides at a time) called codons, from the 5' end to the 3' end. the 5' end to the 3' end.

Each amino acid is specified by the mRNA's Each amino acid is specified by the mRNA's codon, and then pairs with a sequence of three codon, and then pairs with a sequence of three complementary nucleotides carried by a complementary nucleotides carried by a particular tRNA (anticodon).particular tRNA (anticodon).

Genetic codeGenetic code Since RNA is constructed from four types of nucleotides.Since RNA is constructed from four types of nucleotides.

There are 64 possible triplet sequences or codons (4x4x4).There are 64 possible triplet sequences or codons (4x4x4).

Three of these possible codons specify the termination of the Three of these possible codons specify the termination of the polypeptide chain. polypeptide chain.

They are called "They are called "stop codonsstop codons". That leaves 61 codons to ". That leaves 61 codons to specify only 20 different amino acids. specify only 20 different amino acids.

Characteristics of the genetic Characteristics of the genetic codecode

1.1. DegenerateDegenerate: 64 codons: 64 codons1.1. (AUG(AUG initiator) initiator)

2.2. AGA, AGG, UGAAGA, AGG, UGAterminatorterminatorTher remains 61 coding codonsTher remains 61 coding codons

2.2. UnambigousUnambigous: every codon codes for a single aa: every codon codes for a single aa

3.3. Non overlappingNon overlapping

4.4. Non punctuatedNon punctuated

5.5. UniversalUniversal

Central Dogma of Molecular Biology

DNA mRNA

PermanentGeneticarchive

Same in all cells

transcription

translation

rev transcription

Protein

Protein synthesis is achieved by the Protein synthesis is achieved by the interaction of mainly the following interaction of mainly the following molecules (nucleus and cytoplasm)molecules (nucleus and cytoplasm) DNADNA RNA: mRNA and tRNARNA: mRNA and tRNA Several enzymes Several enzymes RibosomesRibosomes Amino acidsAmino acids

Key Components in the Key Components in the Process of Protein SynthesisProcess of Protein Synthesis

DNADNA: : Stores all the genetic information of an Stores all the genetic information of an

organism. Same in all cells of an organismorganism. Same in all cells of an organism Determines which protein to make and Determines which protein to make and

when to make it. when to make it. Only a gene that is to be expressed is Only a gene that is to be expressed is

transcribed to mRNAtranscribed to mRNA



RNAs: mRNARNAs: mRNA mRNA carries genetic information from mRNA carries genetic information from

DNA segment (a gene), to the translation DNA segment (a gene), to the translation site, i.e. ribosome in the cytoplasm. site, i.e. ribosome in the cytoplasm.

mRNA for a gene is transiently present in mRNA for a gene is transiently present in the cell when that gene is expressed, the cell when that gene is expressed, therefore different in different cellstherefore different in different cells

tRNAtRNA brings amino acids to the brings amino acids to the translation site, the ribosome.translation site, the ribosome.

EnzymesEnzymes RNA polymerase-RNA polymerase-formation of RNA from a formation of RNA from a

DNA templateDNA template DNA polymeraseDNA polymerase-DNA replication-DNA replication Reverse transcriptase-Reverse transcriptase-formation of DNA using formation of DNA using

an RNA template an RNA template Synthetases-Synthetases-catalyse covalent links by catalyse covalent links by

activation of the tRNA. There is (close to) one activation of the tRNA. There is (close to) one synthetase per amino acidsynthetase per amino acid


RibosomesRibosomes They provide the sites for protein They provide the sites for protein

synthesissynthesis Amino acidsAmino acids

These are the building blocks of These are the building blocks of proteins. They are sequentially joined proteins. They are sequentially joined together to form polypeptide chainstogether to form polypeptide chains


The basic steps of translation areThe basic steps of translation are

InitiationInitiation ElongationElongation TerminationTermination

TranslationTranslation(cytoplasm)(cytoplasm)


InitiationInitiation Special tRNAi, charged with methionine (Met-Special tRNAi, charged with methionine (Met-

tRNAi)tRNAi) is used to initiate translation (AUG)is used to initiate translation (AUG)

Eukaryotic Initiation Factor 2 (eIF-2) plus GTP Eukaryotic Initiation Factor 2 (eIF-2) plus GTP binds Met-tRNAi, the complex enters the P site of binds Met-tRNAi, the complex enters the P site of the 40S ribosomethe 40S ribosome..

eIF-3 facilitates the binding of ‘Met-tRNAi-40S eIF-3 facilitates the binding of ‘Met-tRNAi-40S ribosome complex’ to mRNA by migrating the ribosome complex’ to mRNA by migrating the complexcomplex along mRNA to AUG start codon (ATP along mRNA to AUG start codon (ATP used for energy)used for energy)

InitiationInitiation eIF-6 brings eIF-6 brings 60S60S ribosome to ribosome to Met-tRNAi-Met-tRNAi-

40S-mRNA complex40S-mRNA complex, , and eIFs-2 and 3 are and eIFs-2 and 3 are releasedreleased

When 60S-40S joining is complete, all eIFs When 60S-40S joining is complete, all eIFs dissociatedissociate

Met-tRNAi-40S-60S-mRNA Met-tRNAi-40S-60S-mRNA complex complex (polysome) left(polysome) left


ElongationElongation Each amino acid has its activating enzyme Each amino acid has its activating enzyme

that activates and catalyses its transfer to a that activates and catalyses its transfer to a molecule of aminoacyl-tRNA.molecule of aminoacyl-tRNA.

tRNA binds amino acid (by covalent bonds) tRNA binds amino acid (by covalent bonds) in presence of in presence of Aminoacyl-tRNA Aminoacyl-tRNA synthetase, synthetase, to yield a reactive to yield a reactive Aminoacyl-Aminoacyl-tRNAtRNA


ElongationElongation Aminoacyl-tRNA then moves to the Aminoacyl-tRNA then moves to the

polysomepolysome

Anticodon of the Aminoacyl-tRNA Anticodon of the Aminoacyl-tRNA recognizes and binds the mRNA codon recognizes and binds the mRNA codon (attached onto the polysome) by H-bonds(attached onto the polysome) by H-bonds


ElongationElongation Amino acids are transferred from the tRNA Amino acids are transferred from the tRNA

to the growing chain under to the growing chain under Peptidyl Peptidyl transferase transferase enzyme enzyme

The Polysome moves along mRNA, 5’-The Polysome moves along mRNA, 5’-3’ synthesizing polypeptide chain, 1 3’ synthesizing polypeptide chain, 1 amino acid per charged tRNA, N- to C-amino acid per charged tRNA, N- to C-terminal.terminal.

Amino acid order is directed by the Amino acid order is directed by the nucleotide sequence on mRNAnucleotide sequence on mRNA


TerminationTermination Process not fully understoodProcess not fully understood

Stop (nonsense) codonsStop (nonsense) codons UAG (amber),UAG (amber), UAA (ochre),UAA (ochre), UGA (opal)UGA (opal)

recognized by a eukaryotic release factor (eRF)recognized by a eukaryotic release factor (eRF) Polypeptide released from last tRNA Polypeptide released from last tRNA Expulsion of tRNA, dissociation of ribosome, Expulsion of tRNA, dissociation of ribosome,

as separate subunitsas separate subunits


Post-ribosomal processingPost-ribosomal processing

In specialized tissues in eukaryotesIn specialized tissues in eukaryotes Examples: Examples:

hydroxylation of proline and lysine in collagenhydroxylation of proline and lysine in collagen addition of carbohydrate groupsaddition of carbohydrate groups

glycoproteinsglycoproteins removal of a peptide: activation of insulin and removal of a peptide: activation of insulin and

proteolytic enzymesproteolytic enzymes

The Protein MoleculeThe Protein Molecule

Sickle cell mutataion

Heme

Iron atom

Sickle cell mutataion

Heme

Iron atom

Hemoglobin

FinalFinal

The process is extremely fastThe process is extremely fast The process is complexThe process is complex The process is very crucial to The process is very crucial to

lifelife

Recombinant DNA Technology and Recombinant DNA Technology and Gene CloningGene Cloning

Introduction Introduction

1.1. Recombinant DNARecombinant DNA Is DNA that has been made artificially by joining Is DNA that has been made artificially by joining

two/more DNAs from different sources.two/more DNAs from different sources.Its gene product is called a recombinant proteinIts gene product is called a recombinant protein

2. 2. Recombinant DNA TechnologyRecombinant DNA TechnologyIs the technology/technique of joining 2 or Is the technology/technique of joining 2 or more DNA segments to obtain a single more DNA segments to obtain a single recombinant DNA moleculerecombinant DNA molecule

Background and UseBackground and Use

Recombinant DNA technology came into Recombinant DNA technology came into being in the 1970sbeing in the 1970s

It is a powerful toll that has enabled science to It is a powerful toll that has enabled science to manipulate DNA/genes to suit human manipulate DNA/genes to suit human requirementsrequirements Express a desired gene to get a recombinant Express a desired gene to get a recombinant

protein for studyprotein for study Create gene librariesCreate gene libraries

Tools for Recombinant DNA Tools for Recombinant DNA TechnologyTechnology

The technology requires that DNA is The technology requires that DNA is cutcut, and , and joinedjoined with other fragments of DNA to get the with other fragments of DNA to get the new (Recombinant) DNAnew (Recombinant) DNA

CuttingCutting: Restriction (endonucleases) enzymes: Restriction (endonucleases) enzymes JoiningJoining: Ligases : Ligases

A restriction enzyme recognizes and cuts DNA A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. only at a particular sequence of nucleotides.

E.g the bacterium E.g the bacterium Hemophilus aegypticusHemophilus aegypticus produces an enzyme named produces an enzyme named HaeHaeIIIIII that cuts that cuts DNA wherever it encounters the sequence DNA wherever it encounters the sequence 5'GGCC3'5'GGCC3'3'CCGG5'3'CCGG5'

1. Restriction enzymes1. Restriction enzymes

The cut is made between the adjacent G and C.The cut is made between the adjacent G and C. 5'GGCC3‘5'GGCC3‘3'CCGG53'CCGG5

Restriction enzymesRestriction enzymes

Restriction enzymesRestriction enzymes

They cut DNA at specific short sequencesThey cut DNA at specific short sequences

HaeIII and AluI cut straight across the double helix HaeIII and AluI cut straight across the double helix producing producing "blunt""blunt" ends. ends.

However, many restriction enzymes cut in an offset However, many restriction enzymes cut in an offset fashion. fashion.

The ends of the cut have an overhanging piece of The ends of the cut have an overhanging piece of single-stranded DNA. These are called single-stranded DNA. These are called "sticky ends" "sticky ends" because they are able to form base pairs with any because they are able to form base pairs with any DNA molecule that contains the complementary DNA molecule that contains the complementary sticky end. sticky end.

Restriction digestionRestriction digestion

Any other source of DNA treated with the Any other source of DNA treated with the same enzyme will produce such molecules.same enzyme will produce such molecules.

Mixed together, these molecules can join with Mixed together, these molecules can join with each other by the base pairing between their each other by the base pairing between their sticky ends. sticky ends.

The union can be made permanent by another The union can be made permanent by another enzyme, enzyme, DNA ligaseDNA ligase, that forms covalent , that forms covalent bonds along the backbone of each strand. bonds along the backbone of each strand.

The result is a molecule of The result is a molecule of recombinant DNArecombinant DNA ((rDNArDNA).).

Some general features of Restriction Some general features of Restriction enzymes/digestionenzymes/digestion

1. They split both DNA strands1. They split both DNA strands

2. Recognize ‘specific’ sequences,2. Recognize ‘specific’ sequences,

3. the sequence recognized is usually 4-8 np3. the sequence recognized is usually 4-8 np

4. Found in prokaryotes to cleave foreign DNA4. Found in prokaryotes to cleave foreign DNA

5. Most cleave to produce symmetrical fragments5. Most cleave to produce symmetrical fragments

2. Cloning vectors2. Cloning vectors

Are DNA stuff used for insertion and cloning Are DNA stuff used for insertion and cloning of target genesof target genes

Many types exist, but bacterial plasmids have Many types exist, but bacterial plasmids have many advantages over the others!many advantages over the others!

PlasmidsPlasmids

Circular, exrachromosomal DNA in Circular, exrachromosomal DNA in prokaryotesprokaryotes

Have own origin of replication, singleHave own origin of replication, single Mainly for drug resistance in bacteriaMainly for drug resistance in bacteria

Are small (a few thousand base pairs) Are small (a few thousand base pairs)

Usually carry only one or a few genes Usually carry only one or a few genes

PlasmidsPlasmids

Plasmids used in genetic engineering are Plasmids used in genetic engineering are called vectors. called vectors.

They are used to transfer genes from one They are used to transfer genes from one organism to another and typically contain a organism to another and typically contain a genetic marker conferring a phenotype that can genetic marker conferring a phenotype that can be selected for or against. be selected for or against.

TransformationTransformation

Plasmids enter the bacterial cell with relative Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in for the rapid spread of antibiotic resistance in hospitals and elsewhere. hospitals and elsewhere.

Plasmids can be deliberately introduced into Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell bacteria in the laboratory transforming the cell with the incoming genes. with the incoming genes.

Detection of transformed E.coliDetection of transformed E.coli

An overview of applications of An overview of applications of recombinant DNA technologyrecombinant DNA technology

Name of the Project: To produce human Name of the Project: To produce human lactogenlactogen in in large amounts in the laboratorylarge amounts in the laboratory

Steps:Steps:1. know the gene that encode the lactogen protein in 1. know the gene that encode the lactogen protein in

manman2.Have a suitable cloning vector2.Have a suitable cloning vector3. Digest the cloning vector and human DNA with same 3. Digest the cloning vector and human DNA with same

Restriction enzymeRestriction enzyme4. insert the gene into the cloning vector and ligate by 4. insert the gene into the cloning vector and ligate by

DNA ligaseDNA ligase5. Introduce the recombinant cloning vector into E.coli 5. Introduce the recombinant cloning vector into E.coli

6. culture E.coli6. culture E.coli

NB the E.coli will now produce among others the NB the E.coli will now produce among others the human lactogen protein!human lactogen protein!

For the whole process to be useful, the recombinant For the whole process to be useful, the recombinant molecule must be replicated many times to provide molecule must be replicated many times to provide material for analysis, sequencing, etc. material for analysis, sequencing, etc.

Producing many identical copies of the same Producing many identical copies of the same recombinant molecule is called recombinant molecule is called cloningcloning. Cloning can . Cloning can be done be done in vitroin vitro, by a process called the , by a process called the polymerase polymerase chain reactionchain reaction ( (PCRPCR). ).

In vitroIn vitro cloning of DNA: The cloning of DNA: The Polymerase Chain ReactionPolymerase Chain Reaction

Next?Next?

documentig

Technology

strand of dna

dna template

function of dna

dna segment

dna helicase

mrna transcription

dna promoter region

cytoplasm synthesis