Introduction to Microbial Genetics

Introduction to Microbial Genetics

Microbiology 221Microbiology 221

The Race for the Double HelixThe Race for the Double Helix Rosalind

Franklin and Maurice Wilkins at Kings College

Studied the A and B forms of DNA

Rosalind’s famous x-ray crystallography picture of the B form held the secret, but she didn’t realize its significance

Rosalind Franklin and Maurice Wilkins at Kings College

Studied the A and B forms of DNA

Rosalind’s famous x-ray crystallography picture of the B form held the secret, but she didn’t realize its significance

The Race for the Double HelixThe Race for the Double Helix Watson and Crick

formed an unlikely partnership

A 22 year old PhD and a 34 year old “want to be” PhD

embarked on a model making venture at Cambridge

Used the research of other scientists to determine the nature of the double helix

Watson and Crick formed an unlikely partnership

A 22 year old PhD and a 34 year old “want to be” PhD

embarked on a model making venture at Cambridge

Used the research of other scientists to determine the nature of the double helix

Nucleic Acid CompositionDNA and RNA

Nucleic Acid CompositionDNA and RNA DNA – Basic Molecules

Purines – adenine and guaninePyrmidines – cytosine and thymineSugar – DeoxyribosePhosphate phosphate group

http://www.dnai.org/index.htm -  DNA background

DNA – Basic MoleculesPurines – adenine and guaninePyrmidines – cytosine and thymineSugar – DeoxyribosePhosphate phosphate group

http://www.dnai.org/index.htm -  DNA background

Double HelixDouble Helix Two polynucleotide strands joined by

phosphodiester bonds( backbone) Complementary base pairing in the

center of the moleculeA= T and C G – base pairing.

Two hydrogen bonds between A and T and three hydrogen bonds between C and G.

A purine is bonded to a complementary pyrimidine

Bases are attached to the 1’ C in the sugar

At opposite ends of the strand – one strand has the 3’hydroxyl, the other the 5’ hydroxyl of the sugar molecule

Two polynucleotide strands joined by phosphodiester bonds( backbone)

Complementary base pairing in the center of the molecule

A= T and C G – base pairing. Two hydrogen bonds between A and T and three hydrogen bonds between C and G.

A purine is bonded to a complementary pyrimidine

Bases are attached to the 1’ C in the sugar

At opposite ends of the strand – one strand has the 3’hydroxyl, the other the 5’ hydroxyl of the sugar molecule

DNA StructureDNA Structurehttp://www.johnkyrk.com/DNAanatomy.html - DNA structure

Double helix( continued)Double helix( continued) The double helix is right handed

– the chains turn counter-clockwise.

As the strand turn around each other they form a major and minor groove.

The is a distance of .34nm between each base

The distance between two major grooves is 2.4nm or 10 bases

The diameter of the strand is 2nm

The double helix is right handed – the chains turn counter-clockwise.

As the strand turn around each other they form a major and minor groove.

The is a distance of .34nm between each base

The distance between two major grooves is 2.4nm or 10 bases

The diameter of the strand is 2nm

Complementary Base PairingComplementary Base PairingAdenine

pairs with Thymine

Cytosine pairs with Guanine

Adenine pairs with Thymine

Cytosine pairs with Guanine

The end view of DNAThe end view of DNA This view

shows the double helix and the outer backbone with the bases in the center.

An AT base pair is highlighted in white

This view shows the double helix and the outer backbone with the bases in the center.

An AT base pair is highlighted in white

Double helix and anti-parallelDouble helix and anti-parallelDNA is a directional moleculeThe complementary strands

run in opposite directionsOne strand runs 3’-5’The other strand runs 5’ to 3’( the end of the 5’ has the

phosphates attached, while the 3’ end has a hydroxyl exposed)

DNA is a directional moleculeThe complementary strands

run in opposite directionsOne strand runs 3’-5’The other strand runs 5’ to 3’( the end of the 5’ has the

phosphates attached, while the 3’ end has a hydroxyl exposed)

RNA structureRNA structurePolynucleotide – nucleic

acid - Single stranded molecule that can coil back on itself and produce complementary base-pairing ( t- RNA)

Four bases in RNA are Adenine and Guanine ( purines) and Cytosine and Uracil( pyrimidines)

Sugar – ribosePhosphates

Polynucleotide – nucleic acid - Single stranded molecule that can coil back on itself and produce complementary base-pairing ( t- RNA)

Four bases in RNA are Adenine and Guanine ( purines) and Cytosine and Uracil( pyrimidines)

Sugar – ribosePhosphates

RNARNA Three types of RNA

a. Messengerb. Transferc. Ribosomald. nc- non coding RNA’s

Three types of RNAa. Messengerb. Transferc. Ribosomald. nc- non coding RNA’s

Prokaryote DNAProkaryote DNATightly coiledCoiling maintained by

molecules similar to the coiling in eukaryotes

Circular ds molecule

Tightly coiledCoiling maintained by

molecules similar to the coiling in eukaryotes

Circular ds molecule

Some Special CasesSome Special CasesBorrelia burgdoferi ( Lyme

Disease )has a linear chromosome

Other bacteria have multiple chromosomes

Agrobacterium tumefaciens ( Produces Crown Gall disease in plants) has both circular and linear

Borrelia burgdoferi ( Lyme Disease )has a linear chromosome

Other bacteria have multiple chromosomes

Agrobacterium tumefaciens ( Produces Crown Gall disease in plants) has both circular and linear

Prokaryote chromosomesProkaryote chromosomesCircular DNACircular DNA

E. coli – most often studied in molecular biology of prokaryotes

E. coli – most often studied in molecular biology of prokaryotes The genes of E. coli are located

on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a highly organized structure which fits inside the 1-2 micrometer cell in a format which can still be read by the gene expression machinery.

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a highly organized structure which fits inside the 1-2 micrometer cell in a format which can still be read by the gene expression machinery.

Bacterial DNA and SupercoilingBacterial DNA and Supercoiling Bacterial DNA is supercoiled by

DNA gyrase. Chemical inhibition of gyrase without allowing the cells to reprogram gene expression relaxes supercoiling and expands the nucleoid, suggesting that supercoiling is one of the tools used to compress the genome

Bacterial DNA is supercoiled by DNA gyrase. Chemical inhibition of gyrase without allowing the cells to reprogram gene expression relaxes supercoiling and expands the nucleoid, suggesting that supercoiling is one of the tools used to compress the genome

CoilingCoilingCoiling maintained by

GyraseRelaxation of the coils by

Topoisomerase

Coiling maintained by Gyrase

Relaxation of the coils by Topoisomerase

Nucleosome formationNucleosome formation

DNA is more highly organized in eukaryote cells

The DNA is associated with proteins called histones.( eukaryotes)

These are small basic proteins rich in the amino acids lysine and/or arginine

There are five histones in eukaryote cells, H1, H2A, H2B,H3 and H4.

.

DNA is more highly organized in eukaryote cells

The DNA is associated with proteins called histones.( eukaryotes)

These are small basic proteins rich in the amino acids lysine and/or arginine

There are five histones in eukaryote cells, H1, H2A, H2B,H3 and H4.

.

Chromosome structureChromosome structure

http://www.johnkyrk.com/chromosomestructure.html

http://www.johnkyrk.com/chromosomestructure.html

Eukaryote replicationEukaryote replicationThe nature

of DNA replication was elucidated by Meselson and Stahl

The nature of DNA replication was elucidated by Meselson and Stahl

Meselson and Stahl experimentMeselson and Stahl experiment

1. Grew bacteria in heavy Nitrogen – N-15

2. Transferred bacteria to N-14

3. Before bacteria reproduce in new media, all bacteria contain heavy DNA

4. Samples were taken after one round of replication and two round of replication

Semiconservative replicationSemiconservative replication Each original

strand serves a template or pattern for the replication of the new strand.

The new strand contains one original and a newly synthesized strand

Each original strand serves a template or pattern for the replication of the new strand.

The new strand contains one original and a newly synthesized strand

Eukaryote replicationEukaryote replication Multiple linear chromosomes Each chromosome has more

than one origin of replication Approximately 1400 x as long

as bacterial DNA Multiple replicons on a

chromosome Oris along the length – every 10

to 100 um

Multiple linear chromosomes Each chromosome has more

than one origin of replication Approximately 1400 x as long

as bacterial DNA Multiple replicons on a

chromosome Oris along the length – every 10

to 100 um

Replication forksReplication forks Replication forks and bubbles are

formed. Replication proceeds bidirectionally until the bubbles meet

This shortens the length of time necessary to replicate eukaryote chromosomes

The process of elongation occurs at a speed of 50-100 base pairs/minute as compared to 750 to 1000 base pairs/ minute

http://www.johnkyrk.com/DNAreplication.html

Replication forks and bubbles are formed. Replication proceeds bidirectionally until the bubbles meet

This shortens the length of time necessary to replicate eukaryote chromosomes

The process of elongation occurs at a speed of 50-100 base pairs/minute as compared to 750 to 1000 base pairs/ minute

http://www.johnkyrk.com/DNAreplication.html

The origin of replication and replication forks

The origin of replication and replication forks

Eukaryote replicationEukaryote replication During the S phase, there are 100

replication complexes and each one contains as many as 300 replication forks. These replication complexes are stationary. The DNA threads through these complexes as single strands and emerges as double strands.

During the S phase, there are 100 replication complexes and each one contains as many as 300 replication forks. These replication complexes are stationary. The DNA threads through these complexes as single strands and emerges as double strands.

DNA PolymerasesDNA PolymerasesFourteen DNA

polymerases have been observed in human beings as compared to three in E. coli.

Fourteen DNA polymerases have been observed in human beings as compared to three in E. coli.

Prokaryote ReplicationProkaryote Replication

Bidirectional replicationBidirectional replication There is an

origin of replication

Two replication forks are formed

Replication occurs around the circle until they have opened and copied the entire chromosome

Replicon- contains an origin and is replicated as a unit

There is an origin of replication

Two replication forks are formed

Replication occurs around the circle until they have opened and copied the entire chromosome

Replicon- contains an origin and is replicated as a unit

Ori – Origin of replicationOri – Origin of replication Characteristics used to define

Origins: The position on the DNA at which

replication start points (see right) are found.

A DNA sequence that when added to a non-replicating DNA causes it to replicate.

A DNA sequence whose mutation abolishes replication.

A DNA sequence that in vitro is the binding target for enzyme

Characteristics used to define Origins:

The position on the DNA at which replication start points (see right) are found.

A DNA sequence that when added to a non-replicating DNA causes it to replicate.

A DNA sequence whose mutation abolishes replication.

A DNA sequence that in vitro is the binding target for enzyme

TopoisomerasesTopoisomerasesTopoisomerase

When the double helix of DNA, which is composed of two strands, separates, helicase makes these two strands rotate around each other.

The DnaB protein is the helicase most involved in replication, but the n’ protin may also participate in unwinding.

The single stranded binding proteins SSBP help to keep the strand open

But there is a problem due to the topological reason that the unreplicated part ahead of the replication fork will rotate around its helical axis when the two strands separate at the replication fork

Topoisomerase When the double helix of DNA,

which is composed of two strands, separates, helicase makes these two strands rotate around each other.

The DnaB protein is the helicase most involved in replication, but the n’ protin may also participate in unwinding.

The single stranded binding proteins SSBP help to keep the strand open

But there is a problem due to the topological reason that the unreplicated part ahead of the replication fork will rotate around its helical axis when the two strands separate at the replication fork

Topoisomerase actionTopoisomerase action It causes strong strain in the

helix (1). Thus, it is impossible to unlink the double helical structure of DNA without disrupting the continuity of the strands.

In order to perform unraveling of a "compensating winding up" DNA, enzymes are required (1). Topoisomerase changes the linking number as well as catalyzes the interconversionn of other kinds of topological isomers of DNA (2).

It causes strong strain in the helix (1). Thus, it is impossible to unlink the double helical structure of DNA without disrupting the continuity of the strands.

In order to perform unraveling of a "compensating winding up" DNA, enzymes are required (1). Topoisomerase changes the linking number as well as catalyzes the interconversionn of other kinds of topological isomers of DNA (2).

InitiationInitiation Initiation

a. oriC - origin of chromosomal replicationRecognized by DnaA protein - only recognizes if GATC sites are fully methylatedBinding of DnaA allows DnaB to open complexb. DnaB is the replication helicasec. Strand separation by helicased. SSB (single-stranded binding) protein keeps strands aparte. DNA gyrase - a topoisomerase - puts swivel in DNA which allows strands to rotate and relieve strain of unwinding

Initiationa. oriC - origin of chromosomal replicationRecognized by DnaA protein - only recognizes if GATC sites are fully methylatedBinding of DnaA allows DnaB to open complexb. DnaB is the replication helicasec. Strand separation by helicased. SSB (single-stranded binding) protein keeps strands aparte. DNA gyrase - a topoisomerase - puts swivel in DNA which allows strands to rotate and relieve strain of unwinding

ExplanationExplanation

Recall that DNA double helix is tightly wound structure and that bases lie between the two backbones. If these bases are the template for new strand, how do the appropriate enzymes reach these bases? By the unwinding of the helix.

An enzyme called helicase catalyzes the unwinding of short DNA segments just ahead of the replication fork. The reaction is driven by the hydrolysis of ATP.

Recall that DNA double helix is tightly wound structure and that bases lie between the two backbones. If these bases are the template for new strand, how do the appropriate enzymes reach these bases? By the unwinding of the helix.

An enzyme called helicase catalyzes the unwinding of short DNA segments just ahead of the replication fork. The reaction is driven by the hydrolysis of ATP.

Explanation continuedExplanation continued As soon as duplex is unwound, SSB

(single-stranded binding protein) binds to each of the separated strands to prevent them from base-pairing again. Therefore, the bases are exposed to the replication system.

The unwinding of the duplex would cause the entire DNA molecule to swivel except for the action of a topoisomerase (DNA gyrase) which introduce breaks in the DNA just ahead of the unwinding duplex. These breaks are then rejoined after a few revolutions of the duplex.

As soon as duplex is unwound, SSB (single-stranded binding protein) binds to each of the separated strands to prevent them from base-pairing again. Therefore, the bases are exposed to the replication system.

The unwinding of the duplex would cause the entire DNA molecule to swivel except for the action of a topoisomerase (DNA gyrase) which introduce breaks in the DNA just ahead of the unwinding duplex. These breaks are then rejoined after a few revolutions of the duplex.

The need for a primerThe need for a primer

When DNA template is exposed, DNA synthesis must begin. But DNA polymerases not only need a template but also a primer for replication to proceed. Where does the primer come from?

After observations that RNA synthesis is required for DNA synthesis, it was discovered that the synthesis of DNA fragments requires a short length of RNA as a primer.Primosome (complex of 20 polypeptides) makes RNA primers in E. coli

When DNA template is exposed, DNA synthesis must begin. But DNA polymerases not only need a template but also a primer for replication to proceed. Where does the primer come from?

After observations that RNA synthesis is required for DNA synthesis, it was discovered that the synthesis of DNA fragments requires a short length of RNA as a primer.Primosome (complex of 20 polypeptides) makes RNA primers in E. coli

Formation of the PrimerFormation of the Primer Primosome contains primase Primosome moves along DNA duplex in

3'>5' direction (with respect to lagging strand; follows replication fork) even though primer is made in 5'>3' direction(Note: The symbol ">" indicates the direction; that is, the primer is made from 5' to 3'.)n' protein removes SSB in front of primosome

DnaB protein organizes some components of primosome and prepares DNA for primasePrimase forms the primer

Primosome contains primase Primosome moves along DNA duplex in

3'>5' direction (with respect to lagging strand; follows replication fork) even though primer is made in 5'>3' direction(Note: The symbol ">" indicates the direction; that is, the primer is made from 5' to 3'.)n' protein removes SSB in front of primosome

DnaB protein organizes some components of primosome and prepares DNA for primasePrimase forms the primer

DNA POLYMERASE IIIDNA POLYMERASE III Holoenzyme Complex that

synthesizes most of the DNA copy contains the DNA polymerase enzyme and other proteins

The gamma delta complex and the B subunits of the holoenzyme bind it to the template and the primer

The alpha subunit carries out the actual polymerization reaction

All of the proteins form a huge complex called the replisome

Holoenzyme Complex that

synthesizes most of the DNA copy contains the DNA polymerase enzyme and other proteins

The gamma delta complex and the B subunits of the holoenzyme bind it to the template and the primer

The alpha subunit carries out the actual polymerization reaction

All of the proteins form a huge complex called the replisome

DNA polymerase IIIDNA polymerase IIIThis is a

stationary complex that probably attached to the plasma membrane.

The DNA moves through the replisome and is copied

This is a stationary complex that probably attached to the plasma membrane.

The DNA moves through the replisome and is copied

Elongation of the chainElongation of the chain

dCTP dCMP +

PPiEnergy is

supplied for biosynthesis by the cleaving of the phosphate bond

dCTP dCMP +

PPiEnergy is

supplied for biosynthesis by the cleaving of the phosphate bond

Elongation( continued)Elongation( continued)Elongation proceeds in 5' >

3' direction and requires 1) all 4 deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP, dTTP), 2) Mg+ ions, 3) a primer made of nucleic acid, and 4) a DNA template.

Rate of elongation = 750 - 1000 nucleotides per secondRate of formation of initiation complex = 1-2 minutes

Elongation proceeds in 5' > 3' direction and requires 1) all 4 deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP, dTTP), 2) Mg+ ions, 3) a primer made of nucleic acid, and 4) a DNA template.

Rate of elongation = 750 - 1000 nucleotides per secondRate of formation of initiation complex = 1-2 minutes

ElongationElongation Elongation

DNA polymerase I, II and III in E .coliDNA polymerase III holoenzyme - complex of 7 polypeptides

Replisome - primosome and 2 DNA polymerase III - synthesizes DNA on both strands simultaneously without dissociating from DNA

DNA polymerase III catalyzes the addition of deoxyribonucleotide units to end of the DNA strand with release of inorganic pyrophosphate (PPi)(DNA)n residues + dNTP < > (DNA)n + 1 residues + PPiAttachment of new units is by their a-phosphate groups to a free 3'-hydroxyl end of preexisting DNA chain.

ElongationDNA polymerase I, II and III in E .coliDNA polymerase III holoenzyme - complex of 7 polypeptides

Replisome - primosome and 2 DNA polymerase III - synthesizes DNA on both strands simultaneously without dissociating from DNA

DNA polymerase III catalyzes the addition of deoxyribonucleotide units to end of the DNA strand with release of inorganic pyrophosphate (PPi)(DNA)n residues + dNTP < > (DNA)n + 1 residues + PPiAttachment of new units is by their a-phosphate groups to a free 3'-hydroxyl end of preexisting DNA chain.

The lagging strand and discontinuous replication

The lagging strand and discontinuous replication The replication on the 5’ to 3’

strand differs The template strand still must

be read from 3’ to 5’ The reading begins at the

replication fork Occurs at the same time as the

synthesis of the lagging strand Same steps in synthesis of DNA But DNA is synthesized in

pieces about 1000 to 2000 bases in length. These are known as Okazaki fragments

The replication on the 5’ to 3’ strand differs

The template strand still must be read from 3’ to 5’

The reading begins at the replication fork

Occurs at the same time as the synthesis of the lagging strand

Same steps in synthesis of DNA But DNA is synthesized in

pieces about 1000 to 2000 bases in length. These are known as Okazaki fragments

Okazaki fragmentsOkazaki fragments After the lagging strand has been

duplicated by the formation of Okazaki fragments, DNA Polymerase I or RNase H removes the RNA primer. Polymerase I synthesizes the complementary DNA to fill the gap resulting from the RNA delection.

The polymerase removes one nucleotide at a time and then replaces it

AMP( RNA nucleotide) replaced by dAMP( DNA nucleotide)

After the lagging strand has been duplicated by the formation of Okazaki fragments, DNA Polymerase I or RNase H removes the RNA primer. Polymerase I synthesizes the complementary DNA to fill the gap resulting from the RNA delection.

The polymerase removes one nucleotide at a time and then replaces it

AMP( RNA nucleotide) replaced by dAMP( DNA nucleotide)

DNA ligaseDNA ligase Ligase can catalyze

the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate.

This can fill in the unattached gap left when the RNA primer is removed and filled in.

The DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.

Ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate.

This can fill in the unattached gap left when the RNA primer is removed and filled in.

The DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.

The End of ReplicationThe End of Replication DNA replication stops when the

polymerase complex reaches a termination site on the DNA in E. coli

The Tus protein binds to the ter site and halts replication.

In many prokaryotes the replication process stops when the replication forks meet

DNA replication stops when the polymerase complex reaches a termination site on the DNA in E. coli

The Tus protein binds to the ter site and halts replication.

In many prokaryotes the replication process stops when the replication forks meet

Plasmid replicationPlasmid replication ColE1 is a naturally occurring plasmid of E.

coli. Its replication is controlled independently of the replication of the host chromosome.

Two plasmids with the same origin of replication can not coexist in the same cell.

The ColE1 origin, defined by molecular genetic methods, is in a region from which two RNAs are transcribed.

An active RNase H gene is required for ColE1 replication. RNase H cleaves the RNA II transcript. The remaining RNA serves as primer for initiation of replication.

RNA I binds to 5' sequences of RNA II via pseudoknots and regular complementary pairing. This binding is stabilized by the ROP or ROM protein.

The binding prevents changes in the conformation of RNA II that would otherwise result in RNAse H cleavage.

ColE1 is a naturally occurring plasmid of E. coli. Its replication is controlled independently of the replication of the host chromosome.

Two plasmids with the same origin of replication can not coexist in the same cell.

The ColE1 origin, defined by molecular genetic methods, is in a region from which two RNAs are transcribed.

An active RNase H gene is required for ColE1 replication. RNase H cleaves the RNA II transcript. The remaining RNA serves as primer for initiation of replication.

RNA I binds to 5' sequences of RNA II via pseudoknots and regular complementary pairing. This binding is stabilized by the ROP or ROM protein.

The binding prevents changes in the conformation of RNA II that would otherwise result in RNAse H cleavage.

Rolling Circle Replication – Occurs in Conjugation in E. coli.

How can one account for the high fidelity of replication?How can one account for the high fidelity of replication?

The answer is based on the fact that DNA Polymerase absolutely requires 3'-OH end of base-paired primer strand on which to add new nucleotides.

DNA polymerase III has 3' > 5' exonuclease activity. It was discovered that DNA polymerase III actually proofreads the newly synthesized strand before continuing with replication. When incorrect nucleotide is incorporated, DNA polymerase III, by means of the 3' > 5' exonuclease activity, "backs up" and hydrolyzes off the incorrect nucleotide. The correct nucleotide is then added to the chain and elongation is resumed.

All 3 DNA polymerases have 3'>5' exonuclease activity

Proofreading ability - 1 error in 10 million

The answer is based on the fact that DNA Polymerase absolutely requires 3'-OH end of base-paired primer strand on which to add new nucleotides.

DNA polymerase III has 3' > 5' exonuclease activity. It was discovered that DNA polymerase III actually proofreads the newly synthesized strand before continuing with replication. When incorrect nucleotide is incorporated, DNA polymerase III, by means of the 3' > 5' exonuclease activity, "backs up" and hydrolyzes off the incorrect nucleotide. The correct nucleotide is then added to the chain and elongation is resumed.

All 3 DNA polymerases have 3'>5' exonuclease activity

Proofreading ability - 1 error in 10 million

Exonucleases and repairExonucleases and repair DNA polymerase I also has

5'>3' exonuclease activity which removes RNA primer and 5'>3' polymerase activity which fills in the gap

This causes a single-stranded break in the DNA - called a nickDNA ligase repairs nick by creating a phosphodiester bond

DNA polymerase I also has 5'>3' exonuclease activity which removes RNA primer and 5'>3' polymerase activity which fills in the gap

This causes a single-stranded break in the DNA - called a nickDNA ligase repairs nick by creating a phosphodiester bond

Genes and Gene ExpressionGenes and Gene Expression Genes are written in a code consisting of

groups of three letters called triplets. There are four letters in the DNA alphabet.

There are 64 possible arrangements of the four letters in groups of three

The triplets specify amino acids for the synthesis of proteins from the information contained in the gene

Genes can also specify t- RNA or r- RNAs The gene begins with a start triplet and ends

with a stop. The bases between the start and the stop are called an open reading frame, ORF.

The information in the gene is transcribed by RNA polymerase.

It reads the gene from 3’ to 5’ The template strand is now referred to as the

CRICK strand and the nontemplate strand is now known as the WATSON strand

DNA sequences are stored in data bases as the WATSON strandReference - COLD SPRING HARBOR - 2003

Genes are written in a code consisting of groups of three letters called triplets.

There are four letters in the DNA alphabet. There are 64 possible arrangements of the four letters in groups of three

The triplets specify amino acids for the synthesis of proteins from the information contained in the gene

Genes can also specify t- RNA or r- RNAs The gene begins with a start triplet and ends

with a stop. The bases between the start and the stop are called an open reading frame, ORF.

The information in the gene is transcribed by RNA polymerase.

It reads the gene from 3’ to 5’ The template strand is now referred to as the

CRICK strand and the nontemplate strand is now known as the WATSON strand

DNA sequences are stored in data bases as the WATSON strandReference - COLD SPRING HARBOR - 2003

Promoters are at the beginning of the GenePromoters are at the beginning of the Gene RNA polymerase recognizes a binding

site in front of the gene. This is referred to as upstream of the gene.

The direction of transcription is referred to as downstream

Different genes have different promoters. IN E. coli the promoters have two functions

The RNA recognition site for transcription which is the consensus sequence for prokaryotes is

5’ TTGACA3’ ( Watson strand) which means on the reading strand 3’ AACTGT5’ ( Crick strand)

RNA polymerase recognizes a binding site in front of the gene. This is referred to as upstream of the gene.

The direction of transcription is referred to as downstream

Different genes have different promoters. IN E. coli the promoters have two functions

The RNA recognition site for transcription which is the consensus sequence for prokaryotes is

5’ TTGACA3’ ( Watson strand) which means on the reading strand 3’ AACTGT5’ ( Crick strand)

The Pribnow Box and Shane -DalgarnoThe Pribnow Box and Shane -Dalgarno The RNA binding site has a consensus

sequence of 5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+) This is where the DNA begins to

become unwound for transcription The initially transcribed sequence of

the gene may not reflect doing but may be a leader sequence.

The prokaryotes usually contain a consensus sequence known as the Shane Delgarno which is complememtary to the 16s rRNA on the ribosome

( small subunit ) The leader sequence also may

regulate transcription

The RNA binding site has a consensus sequence of

5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+) This is where the DNA begins to

become unwound for transcription The initially transcribed sequence of

the gene may not reflect doing but may be a leader sequence.

The prokaryotes usually contain a consensus sequence known as the Shane Delgarno which is complememtary to the 16s rRNA on the ribosome

( small subunit ) The leader sequence also may

regulate transcription

The structure of a prokaryote geneThe structure of a prokaryote gene

Prokaryote Genes are Prokaryote Genes are Continuous They do not contain introns like

eukaryote genes The gene consists of codons

that will determine the sequence of amino acids in the protein

At the end of the gene there is a terminator sequence rather than an actual stop

The terminator may be at the end of a trailer sequence located downstream from the actual coding region of the gene

Continuous They do not contain introns like

eukaryote genes The gene consists of codons

that will determine the sequence of amino acids in the protein

At the end of the gene there is a terminator sequence rather than an actual stop

The terminator may be at the end of a trailer sequence located downstream from the actual coding region of the gene

The Gene begins withThe Gene begins withDNA is read 3’ to 5’ and m

RNA is synthesized 5’ to 3’3’ TAC is the start tripletThis produces a

complementary mRNA message 5’ AUG 3’ –

Groups of three bases in the messenger RNA formed are referred to as CODONS

DNA is read 3’ to 5’ and m RNA is synthesized 5’ to 3’

3’ TAC is the start tripletThis produces a

complementary mRNA message 5’ AUG 3’ –

Groups of three bases in the messenger RNA formed are referred to as CODONS

RNA POLYMERASE

Wobble

•There is wobble in the DNA code – This is a protection from mutations

•More than one codon can specify the same amino acid

• Note arginine - CGU, CGC,CGA, CGG all code for arginine – only the third base in the codon changes

•There are two additional codons for arginine as well AGA and AGG these reflect the degenerate nature of the code

Codon chartCodon chart

Genes for t RNAs and r RNAsGenes for t RNAs and r RNAsThe genes for t RNAs have

a promoter and transcribed leader and trailer sequence that are removed prior to their utilization in translation. Genes coding for tRNA may code for more than a single tRNA molecule

The segments coding for r RNAs are separated by spacer sequencs that are removed after transcription.

The genes for t RNAs have a promoter and transcribed leader and trailer sequence that are removed prior to their utilization in translation. Genes coding for tRNA may code for more than a single tRNA molecule

The segments coding for r RNAs are separated by spacer sequencs that are removed after transcription.

t-RNAt-RNA The acceptor stem

includes the 5' and 3' ends of the tRNA.

The 5' end is generated by RNase P

The 3' end is the site which is charged with amino acids for translation.

Aminoacyl tRNA synthetases interact with both the acceptor 3' end and the anticodon when charging tRNAs.

The anticodon matches the codon on mRNA and is read

3’ to 5’

The acceptor stem includes the 5' and 3' ends of the tRNA.

The 5' end is generated by RNase P

The 3' end is the site which is charged with amino acids for translation.

Aminoacyl tRNA synthetases interact with both the acceptor 3' end and the anticodon when charging tRNAs.

The anticodon matches the codon on mRNA and is read

3’ to 5’

t- RNAt- RNAFound in the cytoplasmAmino acyl t- RNA

synthetase is an enzyme that enables the amino acid to attach to t-RNA

Also activates the t- RNAClover leaf has a stem for

attachment to the amino acid and an anticodon on the bottom of the clover leaf

Found in the cytoplasmAmino acyl t- RNA

synthetase is an enzyme that enables the amino acid to attach to t-RNA

Also activates the t- RNAClover leaf has a stem for

attachment to the amino acid and an anticodon on the bottom of the clover leaf

t- RNAt- RNACommon Features a CCA

trinucleotide at the 3' end, unpaired

four base-paired stems, and

One loop containing a T-pseudoU-C sequence and another containing dihydroU.

Common Features a CCA

trinucleotide at the 3' end, unpaired

four base-paired stems, and

One loop containing a T-pseudoU-C sequence and another containing dihydroU.

tRNAtRNA tRNAs attach to

a specific amino acid and carry it to the ribosome

There are 20 amino acids

61 different codons for these amino acids and 61 tRNAs

The anticodon is complementary to the codon

Binds to the codon with hydrogen bonds

tRNAs attach to a specific amino acid and carry it to the ribosome

There are 20 amino acids

61 different codons for these amino acids and 61 tRNAs

The anticodon is complementary to the codon

Binds to the codon with hydrogen bonds

Ribosomal genesRibosomal genes

Very similar to the structure of protein genes

Very similar to the structure of protein genes

tRNA and rRNA genestRNA and rRNA genes The genes for rRNA are also similar to

the organization of genes coding for proteins

All rRNA genes are transcribed as a large precursor molecule that is edited by ribonucleases after transcription to yield the final r RNA products

The genes for rRNA are also similar to the organization of genes coding for proteins

All rRNA genes are transcribed as a large precursor molecule that is edited by ribonucleases after transcription to yield the final r RNA products

Ribosomal RNARibosomal RNACombines with specific

proteins to form ribosomes

Serves as a site for protein synthesis

Associated enzymes and factors control the process of translation

Combines with specific proteins to form ribosomes

Serves as a site for protein synthesis

Associated enzymes and factors control the process of translation

Prokaryote ribosomesProkaryote ribosomes

Ribosomes are small, but complex structures, roughly 20 to 30 nm in diameter, consisting of two unequally sized subunits, referred to as large and small which fit closely together as seen below.

A subunit is composed of a complex between RNA molecules and proteins; each subunit contains at least one ribosomal RNA (rRNA) subunit and a large quantity of ribosomal proteins.

The subunits together contain up to 82 specific proteins assembled in a precise sequence.    

Ribosomes are small, but complex structures, roughly 20 to 30 nm in diameter, consisting of two unequally sized subunits, referred to as large and small which fit closely together as seen below.

A subunit is composed of a complex between RNA molecules and proteins; each subunit contains at least one ribosomal RNA (rRNA) subunit and a large quantity of ribosomal proteins.

The subunits together contain up to 82 specific proteins assembled in a precise sequence.    

Type of rRNA 

Approximate

number of

nucleotides

Subunit Location

16s 1,542 30s

5s 120 50s

23s 2,904 50s

Prokaryote ribosomal RNA

Prokaryote ribosomes – polysomes- the process of translation

Prokaryote ribosomes – polysomes- the process of translation

Prokaryote transcriptionand translation

Prokaryote transcriptionand translationProkaryote transcription

and translation take place in the cytoplasm

All necessary enzymes and molecules are present for the transcription and translation to take place

Prokaryote transcription and translation take place in the cytoplasm

All necessary enzymes and molecules are present for the transcription and translation to take place

TranslationTranslation

A molecule of messenger RNA binds to the 30S ribosome

( small ribosomal unit) at the Shine Dalgarno sequence

This insures the correct orientation for the molecule

The large ribosomal sub unit locks on top

A molecule of messenger RNA binds to the 30S ribosome

( small ribosomal unit) at the Shine Dalgarno sequence

This insures the correct orientation for the molecule

The large ribosomal sub unit locks on top

The Ribosome The Ribosome There are four

significant positions on the ribosome

EPATWhen the 5’ AUG 3’ of

the mRNA is on the P site the t-RNA with the anticodon, 5’UAG3’ forms a temporary bond to begin translation

There are four significant positions on the ribosome

EPATWhen the 5’ AUG 3’ of

the mRNA is on the P site the t-RNA with the anticodon, 5’UAG3’ forms a temporary bond to begin translation

From Gene to polypeptideFrom Gene to polypeptide

E. Coli Gene MapE. Coli Gene Map

Mutations in DNAMutations in DNAMay be characterized by

their genotypic or phenotypic change

Mutations can alter the phenotype of a microorganisms in different ways

Mutations can involve a change in the cellular or colonial morphology

May be characterized by their genotypic or phenotypic change

Mutations can alter the phenotype of a microorganisms in different ways

Mutations can involve a change in the cellular or colonial morphology

Types of MutationsTypes of Mutations Conditional mutations are those

mutations that are expressed only under specific environmental conditions ( temperature)

Biochemical mutations are those that can cause a change in the biochemistry of the cell

( these may inactivate a biochemical pathway)

These mutants are referred to as auxotrophs because they cannot grow on minimal media

Prototrophs are usually wild type strains capable of growing on minimal media

Conditional mutations are those mutations that are expressed only under specific environmental conditions ( temperature)

Biochemical mutations are those that can cause a change in the biochemistry of the cell

( these may inactivate a biochemical pathway)

These mutants are referred to as auxotrophs because they cannot grow on minimal media

Prototrophs are usually wild type strains capable of growing on minimal media

Two types of mutationsTwo types of mutationsSpontaneous mutations –

These occur without a causative agent during replication

Induced mutations are the result of a substance referred to as a mutagen

Cairns reports that a mutant E. coli strain unable to use lactose is able to regain its ability to use the sugar again – should this be referred to as adaptive mutation?

Spontaneous mutations – These occur without a causative agent during replication

Induced mutations are the result of a substance referred to as a mutagen

Cairns reports that a mutant E. coli strain unable to use lactose is able to regain its ability to use the sugar again – should this be referred to as adaptive mutation?

HypermutationHypermutationOne possible explanation is

hypermutationA starving bacterium has

the ability to generate multiple mutations with special mutator genes that enable them to form bacteria with the ability to metabolize lactose

This is an interesting theory still under investigation

One possible explanation is hypermutation

A starving bacterium has the ability to generate multiple mutations with special mutator genes that enable them to form bacteria with the ability to metabolize lactose

This is an interesting theory still under investigation

Spontaneous mutationsSpontaneous mutationsTypes1. A purine substitutes for a purine or

a pyrimidine substitutes of a pyrimidine. This type of mutation is referred ta as a transition. Most of these can be repaired by proofreading mechanisms

2. A pyrimidine substituted for by a purine is referred to as a transversion. These are rarer due to steric problems in the DNA molecule such as pairing purines with purines.

3. Insertions or deletions cause frame shifts – the code shifts over the number of bases inserted or deleted

Types1. A purine substitutes for a purine or

a pyrimidine substitutes of a pyrimidine. This type of mutation is referred ta as a transition. Most of these can be repaired by proofreading mechanisms

2. A pyrimidine substituted for by a purine is referred to as a transversion. These are rarer due to steric problems in the DNA molecule such as pairing purines with purines.

3. Insertions or deletions cause frame shifts – the code shifts over the number of bases inserted or deleted

Mutation TypesMutation Types Erors in

replication due to base tautomerization

AT and CG pairs are formed when keto groups participate in hydrogen bonds

In contrast enol tautomers produce AC and GT base pairing

Erors in replication due to base tautomerization

AT and CG pairs are formed when keto groups participate in hydrogen bonds

In contrast enol tautomers produce AC and GT base pairing

Spontaneous mutations – another cause

Spontaneous mutations – another causeDepurinationA purine nucleotide can

lose its base It will not base pair

normally It will probably lead to a

transition type mutation after the next round of replication.

Cytosine can be deaminated to uracil which can then create a problem

DepurinationA purine nucleotide can

lose its base It will not base pair

normally It will probably lead to a

transition type mutation after the next round of replication.

Cytosine can be deaminated to uracil which can then create a problem

Frame ShiftsFrame Shifts Additions and

deletions change the reading frame.

The hypothetical origin of deletions and insertions may occur during replication

If the new strand slips an insertion or addition may occur

If the parental slips a deletion may occur

Additions and deletions change the reading frame.

The hypothetical origin of deletions and insertions may occur during replication

If the new strand slips an insertion or addition may occur

If the parental slips a deletion may occur

MutagenesisMutagenesis Any agent that

directly damages DNA, alters its chemistry, or interferes with repair mechanisms will induce mutations

a. Base analogsb. Specific

mispairingc. Intercalating

agentsd. Ionizing

radiation

Any agent that directly damages DNA, alters its chemistry, or interferes with repair mechanisms will induce mutations

a. Base analogsb. Specific

mispairingc. Intercalating

agentsd. Ionizing

radiation

Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication.

The expression of mutationsThe expression of mutationsForward mutations – a mutation

from the wild type to a mutant form is called a forward mutation

Reversion-If the organism regains its wild type characteristics through a second mutation

Back mutation – The actual nucleotide sequence is converted back to the original

Suppressor mutation – overcomes the effects of the first mutation

Forward mutations – a mutation from the wild type to a mutant form is called a forward mutation

Reversion-If the organism regains its wild type characteristics through a second mutation

Back mutation – The actual nucleotide sequence is converted back to the original

Suppressor mutation – overcomes the effects of the first mutation

More on mutationsMore on mutationsPoint mutations – caused by

the change in one DNA baseSilent mutations – mutations

can occur which cause no effect – this is due to the degeneracy of the code ( more than one base coding for the same amino acid)

Missense mutation – changes a codon for one amino acid into a codon for another amino acid

Nonsense – In eukaryotes the substitution of a stop into the sequence of a normal gene

Point mutations – caused by the change in one DNA base

Silent mutations – mutations can occur which cause no effect – this is due to the degeneracy of the code ( more than one base coding for the same amino acid)

Missense mutation – changes a codon for one amino acid into a codon for another amino acid

Nonsense – In eukaryotes the substitution of a stop into the sequence of a normal gene

Detection and isolation of mutantsDetection and isolation of mutants Requires a sensitive system Mutations are rare One in about every 107 – 1011

Replica plating is a technique that is used to detect auxotrophs

It distinguishes between wild type and mutants because of their ability to grow in the absence of a particular biosynthetic end product

Replica plating allows plating on minimal media and enriched media from the same master plate

Requires a sensitive system Mutations are rare One in about every 107 – 1011

Replica plating is a technique that is used to detect auxotrophs

It distinguishes between wild type and mutants because of their ability to grow in the absence of a particular biosynthetic end product

Replica plating allows plating on minimal media and enriched media from the same master plate

The selection of auxotorph revertantsThe selection of auxotorph revertants The lysine

auxotrophs ( Lys-) are treated with a mutagen such as nitroquanidine or uv light to produce revertants

The lysine auxotrophs ( Lys-) are treated with a mutagen such as nitroquanidine or uv light to produce revertants

Ames TestAmes TestDeveloped by Bruce

AmesUsed to test for

carcinogensA mutational reversion

assay based upon mutants of Salmonella typhimurium

Developed by Bruce Ames

Used to test for carcinogens

A mutational reversion assay based upon mutants of Salmonella typhimurium

DNA repair mechanismsDNA repair mechanismsType I -Excision repair Corrects damage which causes

distortions in the double helix A repair endonuclease or uvr ABC

endonuclease removes the damaged bases along with some bases on either side of thee lesion

The usual gap is about 12 nucleotides long. It is filled by DNA polymerase and ligase joins the fragments.

This can remove Thymine-Thymine dimers

A special type of repair utilizes glycosylases to remove damaged or unnatural bases yielding the results discussed above

Type I -Excision repair Corrects damage which causes

distortions in the double helix A repair endonuclease or uvr ABC

endonuclease removes the damaged bases along with some bases on either side of thee lesion

The usual gap is about 12 nucleotides long. It is filled by DNA polymerase and ligase joins the fragments.

This can remove Thymine-Thymine dimers

A special type of repair utilizes glycosylases to remove damaged or unnatural bases yielding the results discussed above

Mutations and repairMutations and repairType II – Removal of lesion Thymine dimers and alkylated bases

are often repaired directly Photoreactivation is the repair of

thymine dimers by splitting them apart into separate thymines with the aid of visible light in a photochemical reaction catalyzed by the enzyme photolyase

Light repair-phr gene - codes for deoxyribodipyrimidine photolyase that, with cofactor folic acid, binds in dark to T dimer. When light shines on cell, folic acid absorbs the light and uses the energy to break bond of T dimer; photolyase then falls off DNA

Type II – Removal of lesion Thymine dimers and alkylated bases

are often repaired directly Photoreactivation is the repair of

thymine dimers by splitting them apart into separate thymines with the aid of visible light in a photochemical reaction catalyzed by the enzyme photolyase

Light repair-phr gene - codes for deoxyribodipyrimidine photolyase that, with cofactor folic acid, binds in dark to T dimer. When light shines on cell, folic acid absorbs the light and uses the energy to break bond of T dimer; photolyase then falls off DNA

Dark repair of mutationsDark repair of mutations Dark repair

Three types1) UV Damage Repair (also called NER - nucleotide excision repair)Excinuclease (an endonuclease; also called correndonuclease [correction endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer (composed of subunits coded by uvrA, uvrB and uvrC genes). UvrA protein and ATP bind to DNA at the distortion. UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA. UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer. UvrD (DNA helicase II; same as DnaB used during replication initiation) separates strands to release 12-bp segment. DNA polymerase I now fills in gap in 5'>3' direction and ligase seals.

Dark repairThree types1) UV Damage Repair (also called NER - nucleotide excision repair)Excinuclease (an endonuclease; also called correndonuclease [correction endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer (composed of subunits coded by uvrA, uvrB and uvrC genes). UvrA protein and ATP bind to DNA at the distortion. UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA. UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer. UvrD (DNA helicase II; same as DnaB used during replication initiation) separates strands to release 12-bp segment. DNA polymerase I now fills in gap in 5'>3' direction and ligase seals.

The Effects of uv lightThe Effects of uv light

Post replication repairPost replication repair If T dimer not repaired, DNA Pol III can't

make complementary strand during replication. Postdimer initiation - skips over lesion and leaves large gap (800 bases). Gap may be repaired by enzymes in recombination system - lesion remains but get intact double helix.

Successful post replication depends upon the ability to recognize the old and newly replicated DNA strands

This is possible because the newly replicated DNA strand lack methyl groups on their bases, whereas the older DNA has methyl groups on the bases of both strands.

The DNA repair system cuts out the mismatch from the non- methylated strand

If T dimer not repaired, DNA Pol III can't make complementary strand during replication. Postdimer initiation - skips over lesion and leaves large gap (800 bases). Gap may be repaired by enzymes in recombination system - lesion remains but get intact double helix.

Successful post replication depends upon the ability to recognize the old and newly replicated DNA strands

This is possible because the newly replicated DNA strand lack methyl groups on their bases, whereas the older DNA has methyl groups on the bases of both strands.

The DNA repair system cuts out the mismatch from the non- methylated strand

Recombination repairRecombination repair The DNA repair for which there is no

remaining template is restored RecA protein cuts a piece of template

DNA from a sister molecule and puts it into the gap or uses it to replace a damaged strand

Rec A also participates in a type of inducible repair known as SOS repair.

If the DNA damage is so great that synthesis stops completely leaving many gaps, the Rec A will bind to the gaps and initiate strand exchange.

It takes on a proteolytic funtion that destroys the lexA repressor protein which regulates genes involved in DNA repair and synthesis

The DNA repair for which there is no remaining template is restored

RecA protein cuts a piece of template DNA from a sister molecule and puts it into the gap or uses it to replace a damaged strand

Rec A also participates in a type of inducible repair known as SOS repair.

If the DNA damage is so great that synthesis stops completely leaving many gaps, the Rec A will bind to the gaps and initiate strand exchange.

It takes on a proteolytic funtion that destroys the lexA repressor protein which regulates genes involved in DNA repair and synthesis