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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