Transcription and Transcription and TranslationTranslation

GenesGenes

The structure of a prokaryote The structure of a prokaryote genegene

GeneGene

• Consensus sequencesConsensus sequences• - 10 and -25 upstream from the start- 10 and -25 upstream from the start• 3’ TAC – start or initiator codon3’ TAC – start or initiator codon• Establishes the reading frame – ORF Establishes the reading frame – ORF

– open reading frame– open reading frame• Bases grouped in 3’s triplet codeBases grouped in 3’s triplet code• Proceeds to stop ( ATT , ATC, ACT or Proceeds to stop ( ATT , ATC, ACT or

termination sequence in prokaryotestermination sequence in prokaryotes

The Pribnow Box and Shane -DalgarnoThe Pribnow Box and Shane -Dalgarno

• The RNA binding site has a consensus sequence ofThe RNA binding site has a consensus sequence of 5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+)5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+)• This is where the DNA begins to become unwound This is where the DNA begins to become unwound

for transcriptionfor transcription• The initially transcribed sequence of the gene may The initially transcribed sequence of the gene may

not reflect doing but may be a leader sequence.not reflect doing but may be a leader sequence.• The prokaryotes usually contain a consensus The prokaryotes usually contain a consensus

sequence known as the Shane Delgarno which is sequence known as the Shane Delgarno which is complememtary to the 16s rRNA on the ribosomecomplememtary to the 16s rRNA on the ribosome

( small subunit )( small subunit )• The leader sequence also may regulate The leader sequence also may regulate

transcriptiontranscription

Promoters are at the beginning of the GenePromoters are at the beginning of the Gene

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

• The direction of transcription is referred to as The direction of transcription is referred to as downstreamdownstream

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

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

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

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

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

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

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

Genes - ContinuedGenes - Continued

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

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

• The information in the gene is transcribed by RNA The information in the gene is transcribed by RNA polymerase.polymerase.

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

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

• DNA sequences are stored in data bases as the DNA sequences are stored in data bases as the WATSON strandWATSON strand

Reference - COLD SPRING HARBOR - 2003Reference - COLD SPRING HARBOR - 2003

Prokaryote Genes areProkaryote Genes are

• ContinuousContinuous• They do not contain introns like eukaryote genesThey do not contain introns like eukaryote genes• The gene consists of codons that will determine The gene consists of codons that will determine

the sequence of amino acids in the proteinthe sequence of amino acids in the protein• At the end of the gene there is a terminator At the end of the gene there is a terminator

sequence rather than an actual stop sequence rather than an actual stop • The terminator may be at the end of a trailer The terminator may be at the end of a trailer

sequence located downstream from the actual sequence located downstream from the actual coding region of the genecoding region of the gene

Transcription BeginsTranscription Begins

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

• 3’ TAC is the start triplet3’ TAC is the start triplet

• This produces a complementary mRNA message This produces a complementary mRNA message 5’ AUG 3’ – 5’ AUG 3’ –

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

RNA POLYMERASE

RNA PolymeraseRNA Polymerase

• There are approximately 2000 There are approximately 2000 molecules of RNA Polymerase per molecules of RNA Polymerase per bacterial cellbacterial cell

• E. coli RNA polymerase is composed of E. coli RNA polymerase is composed of six subunits with a molecular weight of six subunits with a molecular weight of over 400,000 Daover 400,000 Da

RNA POLYMERASERNA POLYMERASE

• Enzyme has been described as a clawEnzyme has been described as a claw

• The Beta portions of the claw form the The Beta portions of the claw form the pincerspincers

• The Alpha portion are on the other end of the The Alpha portion are on the other end of the clawclaw

• The Omega portion is wrapped around the The Omega portion is wrapped around the Beta portionBeta portion

RNA Polymerase ActionRNA Polymerase Action

• Does not require a primer in Does not require a primer in comparison to DNA Polymerasecomparison to DNA Polymerase

• Binds to the promoter sequence and Binds to the promoter sequence and opens the double strands of DNAopens the double strands of DNA

• Builds a chain of RNA by connecting Builds a chain of RNA by connecting the 5’ end of a nucleotide to the 3’ the 5’ end of a nucleotide to the 3’ end of the nucleotide in front of itend of the nucleotide in front of it

LanguageLanguage

• RNA polymerase utilizes the sequence RNA polymerase utilizes the sequence of the template strand in DNA language of the template strand in DNA language to build a complementary copy of RNAto build a complementary copy of RNA

• The strand that is the template is The strand that is the template is referred to as the transcribed strandreferred to as the transcribed strand

• The strand with the same sequence as The strand with the same sequence as RNA with the exception of U instead of RNA with the exception of U instead of T is referred to as the coding strandT is referred to as the coding strand

RNA Polymerase Structure – Core RNA Polymerase Structure – Core enzymeenzyme

Polymerase sub unitsPolymerase sub units

• α2: the two α subunits assemble the enzyme and α2: the two α subunits assemble the enzyme and recognize regulatory factors. recognize regulatory factors.

• These compose the core enzymeThese compose the core enzyme• ββ: this has the polymerase activity (catalyzes the : this has the polymerase activity (catalyzes the

synthesis of RNA) which includes chain initiation and synthesis of RNA) which includes chain initiation and elongation. elongation.

• β': binds to DNA (nonspecifically). β': binds to DNA (nonspecifically). • ω: restores denatured RNA polymerase to its ω: restores denatured RNA polymerase to its

functional form in vitro. It has been observed to offer functional form in vitro. It has been observed to offer a protective/chaperone function to the β' subunit a protective/chaperone function to the β' subunit

RNA Polymerase RNA Polymerase HoloenzymeHoloenzyme

Sigma FactorsSigma Factors

• A A sigma factorsigma factor ( (σ factorσ factor) ) are proteins that are are proteins that are required for the binding of the RNA polymerase to required for the binding of the RNA polymerase to the promoter so that transcription can be initiated. the promoter so that transcription can be initiated. They are also active in the elongation process.They are also active in the elongation process.

Transcription and the Initiation Transcription and the Initiation ComplexComplex

• The core enzyme binds to the The core enzyme binds to the holoenzymeholoenzyme

• This is called promoter recognitionThis is called promoter recognition

• The sigma factors first bins to the -10 The sigma factors first bins to the -10 regionregion

• When the Beta pincer closes around When the Beta pincer closes around the DNAto form the active site channelthe DNAto form the active site channel

around the DNA, this opens the strandsaround the DNA, this opens the strands

RifampinRifampin

• The initiation somplex can be blocked The initiation somplex can be blocked by the antibiotic Rifampinby the antibiotic Rifampin

• Rifampin binds to the RNA Polymerase Rifampin binds to the RNA Polymerase at the active site channel is such a way at the active site channel is such a way that it blocks the elongation of the RNAthat it blocks the elongation of the RNA

Mutations, Antibiotics, and Mutations, Antibiotics, and Antibiotic ResistanceAntibiotic Resistance

Initiation, Elongation, TerminationInitiation, Elongation, Termination

Transcriptional overviewTranscriptional overview

• http://vcell.ndsu.nodak.edu/~christjo/http://vcell.ndsu.nodak.edu/~christjo/vcell/animationSite/transcription/elonvcell/animationSite/transcription/elongation.htmgation.htm

Rho independent( Factor Rho independent( Factor dependent)dependent)

• The termination site consists of two The termination site consists of two sequencessequences

• The first is an inverted repeatThe first is an inverted repeat• When the inverted repeat is transcribed it When the inverted repeat is transcribed it

forms a hairpinforms a hairpin• The inverted repeat is followed by a string of The inverted repeat is followed by a string of

AAAAAAAAAAAsAAAAAAAAAAAs• The RNA molecule will have terminal UUUUsThe RNA molecule will have terminal UUUUs• AU pairs are less stable and this causes the AU pairs are less stable and this causes the

release of the RNA molecule from the DNArelease of the RNA molecule from the DNA

Termination- Factor IndependentTermination- Factor Independent

Factor DependentFactor Dependent

• Rho Dependent ( Rho the most universal Rho Dependent ( Rho the most universal factor in prokaryote cells). There are other factor in prokaryote cells). There are other factorsfactors

• Rho functions in the synthesis of RNAs that Rho functions in the synthesis of RNAs that are not being translatedare not being translated

• Rho forms a ring that binds to a sequence Rho forms a ring that binds to a sequence in the RNA called the rut sitein the RNA called the rut site

• Binds to the RNA and affects the action of Binds to the RNA and affects the action of RNA polymeraseRNA polymerase

Eukaryote Transcription and Eukaryote Transcription and Transcription FactorsTranscription Factors

Genes for t RNAs and r Genes for t RNAs and r RNAsRNAs

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

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

t-RNAt-RNA• The acceptor stem includes The acceptor stem includes

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

• The 5' end is generated by The 5' end is generated by RNase P RNase P

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

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

• The anticodon matches the The anticodon matches the codon on mRNA and is readcodon on mRNA and is read

3’ to 5’3’ to 5’

t- RNAt- RNA

• Found in the cytoplasmFound in the cytoplasm• Amino acyl t- RNA synthetase is an Amino acyl t- RNA synthetase is an

enzyme that enables the amino acid to enzyme that enables the amino acid to attach to t-RNAattach to t-RNA

• Also activates the t- RNAAlso activates the t- RNA• Clover leaf has a stem for attachment Clover leaf has a stem for attachment

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

t- RNAt- RNA

Common FeaturesCommon Features• a CCA trinucleotide at a CCA trinucleotide at

the 3' end, unpairedthe 3' end, unpaired• four base-paired stems, four base-paired stems,

and and • One loop containing a One loop containing a

T-pseudoU-C sequence T-pseudoU-C sequence and another containing and another containing dihydroU. dihydroU.

tRNAtRNA

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

• There are 20 amino There are 20 amino acids acids

• 61 different codons for 61 different codons for these amino acids and these amino acids and 61 tRNAs61 tRNAs

• The anticodon is The anticodon is complementary to the complementary to the codoncodon

• Binds to the codon Binds to the codon with hydrogen bondswith hydrogen bonds

Ribosomal genesRibosomal genes

• Very similar to the structure of Very similar to the structure of protein genesprotein genes

tRNA and rRNA genestRNA and rRNA genes

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

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

Ribosomal RNARibosomal RNA

• Combines with specific proteins to Combines with specific proteins to form ribosomesform ribosomes

• Serves as a site for protein synthesisServes as a site for protein synthesis

• Associated enzymes and factors Associated enzymes and factors control the process of translationcontrol the process of translation

Prokaryote RibosomesProkaryote Ribosomes• Ribosomes are small, but Ribosomes are small, but

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

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

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

30s and 50s Ribosomal 30s and 50s Ribosomal subunitssubunits

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- Prokaryote ribosomes – polysomes- the process of translationthe process of translation

Prokaryote transcriptionProkaryote transcriptionand translationand translation

• Prokaryote transcription and Prokaryote transcription and translation take place in the translation take place in the cytoplasmcytoplasm

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

TranslationTranslation

• A molecule of messenger RNA binds to A molecule of messenger RNA binds to the 30S ribosomethe 30S ribosome

( small ribosomal unit) at the Shine ( small ribosomal unit) at the Shine Dalgarno sequenceDalgarno sequence

• This insures the correct orientation for This insures the correct orientation for the moleculethe molecule

• The large ribosomal sub unit locks on The large ribosomal sub unit locks on toptop

T RNA and Amino acyl t RNA T RNA and Amino acyl t RNA synthetase( Preparation of synthetase( Preparation of tRNAs)tRNAs)• The enzyme amino acyl t RNA The enzyme amino acyl t RNA

Synthetase connects the amino acid Synthetase connects the amino acid to its specific t RNA to form to its specific t RNA to form cognate t cognate t RNARNA

• Hydrolyzes ATP to transfer the amino Hydrolyzes ATP to transfer the amino acid to the 3’OH end of the t RNAacid to the 3’OH end of the t RNA

• The acceptor arm of the t RNA ends The acceptor arm of the t RNA ends in the sequence CCAin the sequence CCA

The RibosomeThe Ribosome

• There are four significant positions on There are four significant positions on the ribosomethe ribosome

• EPATEPAT

• When the 5’ AUG 3’ of the mRNA is on When the 5’ AUG 3’ of the mRNA is on the P site the t-RNA with the anticodon, the P site the t-RNA with the anticodon, 5’UAG3’ forms a temporary bond to 5’UAG3’ forms a temporary bond to begin translationbegin translation

Ribosomal SitesRibosomal Sites

• T site – the 5’ end of the messenger RNA T site – the 5’ end of the messenger RNA enters the ribosomeenters the ribosome

• A site – acceptor site -the relationship of the A site – acceptor site -the relationship of the mRNA to the ribosome is stabilized at this mRNA to the ribosome is stabilized at this site – the amino acylated t RNA is bound to site – the amino acylated t RNA is bound to the mRNAthe mRNA

• P site the peptide bonds are formed between P site the peptide bonds are formed between the amino acids on the t RNAsthe amino acids on the t RNAs

• E site - The mRNA moves to the final position E site - The mRNA moves to the final position on the ribosome as the tRNA is releasedon the ribosome as the tRNA is released

EPATEPAT

Ribosomal structureRibosomal structure

Translation Initiation RegionTranslation Initiation Region

• Should contain the initiator codon( start)Should contain the initiator codon( start)

AUG, may also be GUGAUG, may also be GUG

• This may be at the start of the gene or in anThis may be at the start of the gene or in an

Untranslated leader sequence upstream of Untranslated leader sequence upstream of

Gene ( UTR)Gene ( UTR)

• Shine Delgarno named for two scientists Shine Delgarno named for two scientists who discovered it at -35 binds to the 30s who discovered it at -35 binds to the 30s ribosomal subuit ( 16srRNA)ribosomal subuit ( 16srRNA)

Archaea InitiationArchaea Initiation

• Combination of Eukarya and Bacteria Combination of Eukarya and Bacteria InitiationInitiation

• Archaea and Eukarya have more Archaea and Eukarya have more initiation factors than Bacteriainitiation factors than Bacteria

• Archaea uses formylated methionineArchaea uses formylated methionine

InitiationInitiation

ElongationElongation

ElongationElongation

TranslocaseTranslocase

• Moves the tRNA from the A site to the Moves the tRNA from the A site to the P siteP site

• This displaces the t RNA on the P site This displaces the t RNA on the P site to the E siteto the E site

• Elongation factors function at this Elongation factors function at this pointpoint

• Energy required from the hydrolysis of Energy required from the hydrolysis of GTPGTP

Peptidyl transferasePeptidyl transferase

• Forms the peptide bonds between Forms the peptide bonds between the amino acids attached to the t the amino acids attached to the t RNAsRNAs

• Forms the nascent polypeptide chainForms the nascent polypeptide chain

TerminationTermination

• http://www.phschool.com/science/biohttp://www.phschool.com/science/biology_place/biocoach/translation/term.logy_place/biocoach/translation/term.htmlhtml

• Release factors function in Release factors function in terminationtermination

( RF1 and 3)( RF1 and 3)• GTP is required for energyGTP is required for energy• The termination or stop codon enters The termination or stop codon enters

the A site( UAA, UAG, UGA)the A site( UAA, UAG, UGA)• Recognized by the ribosomeRecognized by the ribosome

TerminationTermination

From Gene to polypeptideFrom Gene to polypeptide

Codon chartCodon chart

•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

WOBBLEWOBBLE

E. Coli Gene MapE. Coli Gene Map

Mutations in DNAMutations in DNA

• May be characterized by their genotypic or May be characterized by their genotypic or phenotypic changephenotypic change

• Mutations can alter the phenotype of a Mutations can alter the phenotype of a microorganisms in different waysmicroorganisms in different ways

• Mutations can involve a change in the Mutations can involve a change in the cellular or colonial morphologycellular or colonial morphology

Types of MutationsTypes of Mutations

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

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

( these may inactivate a biochemical pathway)( these may inactivate a biochemical pathway)

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

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

Two types of mutationsTwo types of mutations

• Spontaneous mutations – These occur without Spontaneous mutations – These occur without a causative agent during replicationa causative agent during replication

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

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

HypermutationHypermutation

• One possible explanation is hypermutationOne possible explanation is hypermutation

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

• This is an interesting theory still under This is an interesting theory still under investigationinvestigation

Spontaneous mutationsSpontaneous mutations

TypesTypes1.1. A purine substitutes for a purine or a pyrimidine A purine substitutes for a purine or a pyrimidine

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

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

3.3. Insertions or deletions cause frame shifts – the Insertions or deletions cause frame shifts – the code shifts over the number of bases inserted or code shifts over the number of bases inserted or deleteddeleted

Mutation TypesMutation Types

• Errors in replication due Errors in replication due to base tautomerizationto base tautomerization

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

• In contrast enol In contrast enol tautomers produce AC tautomers produce AC and GT base pairingand GT base pairing

Spontaneous mutations – Spontaneous mutations – another causeanother cause

• DepurinationDepurination

• A purine nucleotide can lose its baseA purine nucleotide can lose its base

• It will not base pair normallyIt will not base pair normally

• It will probably lead to a transition type It will probably lead to a transition type mutation after the next round of mutation after the next round of replication.replication.

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

Frame ShiftsFrame Shifts

• Additions and deletions Additions and deletions change the reading change the reading frame.frame.

• The hypothetical origin The hypothetical origin of deletions and of deletions and insertions may occur insertions may occur during replicationduring replication

• If the new strand slips an If the new strand slips an insertion or addition may insertion or addition may occuroccur

• If the parental slips a If the parental slips a deletion may occurdeletion may occur

MutagenesisMutagenesis

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

a.a. Base analogsBase analogs

b.b. Specific mispairingSpecific mispairing

c.c. Intercalating agentsIntercalating agents

d.d. Ionizing radiationIonizing 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 mutations

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

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

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

• Suppressor mutation – overcomes the Suppressor mutation – overcomes the effects of the first mutationeffects of the first mutation

More on mutationsMore on mutations

• Point mutations – caused by the change in Point mutations – caused by the change in one DNA baseone DNA base

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

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

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

Detection and isolation of Detection and isolation of mutantsmutants

• Requires a sensitive systemRequires a sensitive system• Mutations are rareMutations are rare• One in about every 10One in about every 1077 – 10 – 101111

• Replica plating is a technique that is used to Replica plating is a technique that is used to detect auxotrophsdetect auxotrophs

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

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

The selection of auxotorph The selection of auxotorph revertantsrevertants

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

Ames TestAmes Test

• Developed by Bruce AmesDeveloped by Bruce Ames

• Used to test for carcinogensUsed to test for carcinogens

• A mutational reversion assay based A mutational reversion assay based upon mutants of upon mutants of

Salmonella typhimuriumSalmonella typhimurium

DNA repair mechanismsDNA repair mechanisms

Type I -Excision repair Type I -Excision repair Corrects damage which causes distortions in the double helixCorrects damage which causes distortions in the double helix• A repair endonuclease or uvr ABC endonuclease removes A repair endonuclease or uvr ABC endonuclease removes

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

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

• This can remove Thymine-Thymine dimersThis can remove Thymine-Thymine dimers• A special type of repair utilizes glycosylases to remove A special type of repair utilizes glycosylases to remove

damaged or unnatural bases yielding the results discussed damaged or unnatural bases yielding the results discussed aboveabove

Mutations and repairMutations and repair

Type II – Removal of lesionType II – Removal of lesion• Thymine dimers and alkylated bases are often Thymine dimers and alkylated bases are often

repaired directlyrepaired directly• Photoreactivation is the repair of thymine dimers by Photoreactivation is the repair of thymine dimers by

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

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

Dark repair of mutationsDark repair of mutations

• Dark repairDark repairThree typesThree types1) UV Damage Repair (also called NER - nucleotide excision 1) UV Damage Repair (also called NER - nucleotide excision repair)repair)Excinuclease (an endonuclease; also called correndonuclease Excinuclease (an endonuclease; also called correndonuclease [correction endo.]) that can detect T dimer, nicks DNA strand on [correction endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer (composed of subunits coded by 5' end of dimer (composed of subunits coded by uvrAuvrA, , uvrBuvrB and and uvrCuvrC genes). genes). UvrA protein and ATP bind to DNA at the distortion. UvrA protein and ATP bind to DNA at the distortion. UvrB binds to the UvrA-DNA complex and increases specificity of UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA. UvrA-ATP complex for irradiated DNA. UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer. of dimer. UvrD (DNA helicase II; same as DnaB used during replication UvrD (DNA helicase II; same as DnaB used during replication initiation) separates strands to release 12-bp segment. initiation) separates strands to release 12-bp segment. DNA polymerase I now fills in gap in 5'>3' direction and ligase DNA polymerase I now fills in gap in 5'>3' direction and ligase seals.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 If T dimer not repaired, DNA Pol III can't make complementary strand during replication. Postdimer complementary strand during replication. Postdimer initiation - skips over lesion and leaves large gap (800 initiation - skips over lesion and leaves large gap (800 bases). Gap may be repaired by enzymes in recombination bases). Gap may be repaired by enzymes in recombination system - lesion remains but get intact double helix. system - lesion remains but get intact double helix.

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

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

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

Recombination repairRecombination repair

• The DNA repair for which there is no remaining template is The DNA repair for which there is no remaining template is restoredrestored

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

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

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

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