Exam #3 Notes Lectures 19-25
Exam #3 Notes Lectures 19-25 MICRB 201
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Exam 3 Notes Lecture 19 Viruses Virus a genetic element that cannot replicate independently of a living host cell Classified by nucleic acids and mode of replication double vs single stranded M as genetic material replicates using DNA dependent DNA polymerase usually double stranded can be single stranded Same as DNA Reverse Transcribing replicates via reverse transcriptase ex Hepadnaviruses the entrie dsDNA genome is transcribed into pregenomic RNA genese are transcribed into mRNAs mRNA are translated into proteins including reverse transcriptase transcriptase transcribes pregenomic RNA into dsDNA Ex Retrovirus ssRNA genomce is reverse transcribed into DNA the DNA is integrated in the hosts cells genomce then undergoes the usual transcription and translation process to express the genes carried by the virus Classified by host s they infect Bacterial bacteriohphages archaeal animal plant other Viron Structure and Organization Viron an extracellular form of a virus exists outside host and facilitates transmission from one host cell to another contains nucleic acid genomce surrounded by a protein usually linear genome multiple shapes and sizes mostly smaller than prokaryotic cells range from 002 to 03 pm smaller genomes than cells higher mutation rates Some virons contain enzymes critical to infection ysozymemakes hole in a cell wall lyses bacterial cell nucleic acid polymerases neuramindiasesenzymes that cleave glycosidic bonds allows liberation of virsuses from cell The capsid is the protein shell that surrounds the genome of a virion Composed of a number of protein molecules arranged in a precise and highly repetitive pattern around the nucleic acid The capsomere is a subunit of the capsid Smallest morphological unit visible with an electron microscope Nucleocapsid the complete complex of nucleic acid and protein packaged in the virion Rodshaped virions display helical symmetry Length of virion is determined by the genome size Width of virion determined by size and packaging of protein subunits Sphericallike virions display icosahedral symmetry Most efficient arrangement of subunits in a closed shell Enveloped viruses have a membrane that surrounds the nucleocapsid Lipid bilayer with embedded proteins Envelope makes initial contact with host cell Quantification of Viruses m number of infectious unit s per volume of uid plaque assay Electron microscopy of vironsvolume can be measured using an electron microscope Intact animal methods measured dose can be given to an individual Lecture 20 Viral Replication Viral cycle attachment penetration synthesis of nucleic acid and protein assembly and packaging lysis Viral Attachment and Penetration The attachment of a virion to its host cell is highly specific It requires complementary receptors on the surface of the virion and its host cell The receptors on the host cell carry out normal functions for the cell eg uptake proteins adhesion cell to cell interaction etc The receptors on the host cell include proteins carbohydrates glycoproteins lipids lipoproteins or complexes a b c The attachment of a virion to its host cell results in changes to both the virion s and cell s surfaces that facilitate penetration h 7 Tail fibers Ex T4 is one the most complex 0 00 39 quoti quot Tail pins penetrationmechanisms Viron I attaches to cell via tail fibers that Outer l 139quot 33 interact with targets on the E coli membrane I t w 7 i 1 Ta outer memebrane Tail fibers retract peptidoglycan J J a J a a d I L J j 4 39 lys39ozym tailtail core makes contact with E coli Cytoplasmic ill membrane ill Vi IIAAI A 1 Q W cell wall lysozymelike enzyme forms a small pore in the peptidoglycan tail sheath contracts viral DNA passes into the cytoplasm Host Cell Protective Mechanisms Host cells possesss mechanisms to protect against virsuses prokaryotesrestriction modification systems CRISPR eukaryotes immune system RNA interferences 1 5 1 Virai DNA iZ lmw if nuc39ease methylase 1 I I CH3 CH3 Baoierial genome Bacteria have restriction enzymes also called restriction endonucleases that cleave dsDNA at specific points into fragments The restriction enzymes recognize short sequences in the DNA Smal CCCGGG GG CCC This prevents infection by effectively destroying the foreign DNA introduced by an infectious agent such as a bacteriophage Given that the sequences recognized by the restriction enzymes are very short the bacterium itself will almost certainly have many of these sequences present in its own DNA In order to prevent destruction of its own DNA by the restriction enzymes the bacterium marks its own DNA by adding methyl groups to it This modification must not interfere with the DNA basepairing and therefore usually only a few specific bases are modified on each strand Viral Nucleic Acid Replication Viruses are divided into two main categories depending on whether their genomes are made of DNA or RNA The mRNA produced by the virus is said to be in the plus configuration its complement is said to be in the minus configuration f quot dsDNA i virus ssDNA dsRNA i ssRNA ssRNA ssRNA Class virus virus VIFUS VIFUS retrovirus Class VII Class II Class III Class IV ClaSS V Class VI Synthesis ofl I V7 70th Strandil Used directly lReverse 1 t jas mRNA Itranscriptionl dsDNA intermediate WSW 39 quot I quot quotF39Wp quot 39 quot of minus strand of minus strand Transcription lof minus strandl I H 7 7 WW V 39 Transcription l swim 3 an dsDNA Intermediate Genome Genome replication Class I classical semiconservative Class II classical semiconservative discard strand Class Vllstranscription followed by reverse transcription DNA VIruses replication Class quotI make ssRNA and transcribe from this to give ssRNA partner Class IV make ssRNA and transcribe from this to give ssRNA genome Class V make ssRNA and transcribe from this to give ssRNA genome Class VI make ssRNA genome by transcription of strand of dsDNA RNA viruses Class I dsDNA viruses The viral dsDNA genome is treated just like the host s dsDNA genome The strand of the viral dsDNA serves as the template for transcription of the viral mRNA using the host s DNAdependent RNA polymerase The viral dsDNA replicates using the host s DNAdependent DNA polymerase Therefore production of progeny requires that the cell be in replication as it is during replication that the cell39s polymerases are active The virus may induce the cell to forcefully undergo cell division which may lead to cancer Examples include Herpesviridae Class II ssDNA viruses The viral ssDNA genome is used as a template to produce a dsDNA intermediate using the host39s DNAdependent DNA polymerase The dsDNA intermediate is treated just like the host s dsDNA genome Used to generate mRNA using the host s DNAdependent RNA polymerase Used to generate more dsDNA using the host s DNAdependent DNA polymerase During packaging into the new virions only the strands of the dsDNA intermediate are packaged Class III dsRNA viruses The strand of the viral dsRNA genome serves as the mRNA so there is no need for the host s DNAdependent RNA polymerase The dsRNA is replicated using a RNAdependent RNA polymerase that was packaged with the infecting virion Class IV Positivesense ssRNA viruses The ssRNA genome serves as the mRNA so there is no need for the host s DNAdependent RNA polymerase The ssRNA is replicated using a RNAdependent RNA polymerase that was packaged with the infecting virion During packaging into the new virions only the ssRNA strands are packaged Class V Negativesense ssRNA viruses The ssRNA genome serves as the template for synthesis of a ssRNA using a RNAdependent RNA polymerase that was packaged with the infecting virion The ssRNA serves as the mRNA During packaging into the new virions only the ssRNA strands are packaged Class VI Positivesense ssRNA viruses that replicate through a DNA intermediate The ssRNA genome is used to produce dsDNA using a reverse transcriptase that was packaged with the infecting virion Reverse transcriptase has sequential biochemical activities RNAdependent DNA polymerase The ssRNA genome is used as the template for the synthesis of ssDNA DNAdependent DNA polymerase The ssDNA is used as the template for the synthesis of dsDNA The dsDNA is treated just like the host s dsDNA genome Used to generate mRNA using the host s DNAdependent RNA polymerase Used to generate more dsDNA using the host s DNAdependent DNA polymerase During packaging into the new virions the mRNA is packaged Class VII dsDNA viruses that replicate through a ssRNA intermediate From the dsDNA mRNA is synthesized as normal using the host s DNA dependent RNA polymerase The mRNA is used to generate dsDNA using a reverse transcriptase that was packaged with the infecting virion Production of Viral Proteins Virus specific proteins must be synthesized in order for the virus to replicate Generation of the virl mRNA occurs first Viral genome serves as the template for the viral mRNA some RNA viruses the viral RNA is the mRNA essential enzymes are contained in the infecting viron proteins synthesized early after infection necessary for replication of the virus nucleic acid late proteins synthesized ie viral coat structural componenets synthesized in large amounts Lecture 21 Viral Diversity Lytic Cycle of Replication The virion attaches to the cell surface The virion injects its genome into the host cell The virial genome is replicated transcribed and translated Many new virions are assembled The host cell is lysed and the new virions are released to infect more cells 91993 Lysogenic Cycle of Replication 1 The virial genome is integrated into and replicates with the host cell s genome 2 This can continue indefinitely 9 Usually happens when the host cell is in nutrient poor conditions 4 Under nutrient poor conditions the resources available to the virus may be limited Moreover the cell density may be very low Therefore it may be in the virus interest to remain dormant Some viruses are capable of both lysogenic and lytic cycles of replication A switch to nutrient rich conditions or a stressor such as UV exposure can trigger the virus to enter the lytic cycle Overview of Bacteriophages Most are dsDNA viruses Most are naked but some possess lipid envelopes They are structurally complex containing heads tails and other components Some bacteriophages like T4 have only a lytic cycle of replication Others like Lamba have both lysogenic and lytic cycles of replication Specific Example of the bacteriophage Lambda Lambda binds to an E coli cell by means of its I protein in the tail tip The I protein interacts with the outer membrane porin LamB involved in the import of the sugar maltose viral genome is injected into the cytoplasm via the mannose permease complex involved in the import of maltose The injected viral genome is linear double stranded DNA However it has single stranded tails cosL and cosR that are each 12 nt long and complementary to each other The sticky tails base pair with each other to yield circular double stranded DNA Switching device determines whether enters lytic or lysogenic cycles The switch involves an intergenic region between two divergently transcribed genes cI and cm The protein cI ultimately activates the expression of genes involved in the lysogenic cycle including a gene coding for an integrase that integrates the Lambda DNA into the E coli chromosome The protein Cro ultimately activates the expression of genes involved in the lytic cycle including a gene that codes for a lytic enzyme that lyses the cell The intergenic region consists of three subregions 0R3 0R2 and 0R1 E coIi s RNA polymerase RNAP can bind to 0R3 and transcribe c E coIi s RNAP can also bind to 0R2 0R1 and transcribe cro At first E coli RNAP binds to both 0R3 and 0R2 0R1 and transcribes both C and cm The protein cI can bind to 0R2 0R1 and prevent transcription of cm by E coIi s RNAP Similarly the protein Cro can bind to 0R3 and prevent transcription of c1 c1 has greater binding affinity for 0R2 0R1 than Cro does for 0R3 All else being equal cI will always outcompete Cro and turn off cro transcription before Cro can turn off cI transcription Thus the default is that c gets transcribed and that cro gets repressed Remember that Cl ultimately activates genes involved in the lysogenic cycle and that Cro ultimately activates genes involved in the lytic cycle Therefore the lysogenic cycle is the default This is what happens in cells in nutrient poor media In cells in nutrient rich media or in cells expressing a stressor various proteases are expressed that are part of normal cellular function cI has evolved to be recognized and cleaved by these proteases The proteases cleave and thereby inactivate cI This enables cm to be transcribed Cro now has the upper hand over Now cro gets transcribed and c1 gets repressed Again remember that Cro ultimately activates genes involved in the lytic cycle and that Cl ultimately activates genes involved in the lysogenic cycle Therefore the lytic cycle is initiated Animal Viruses Entire virion enters the animal cell unlike in prokaryotes Eukaryotic cells contain a nucleus the site of replication for many animal viruses Animal viruses contain all known modes of viral genome replication Many more kinds of enveloped animal viruses than enveloped bacterial viruses exist As animal viruses leave host cell they can remove part of host cell s lipid bilayer for their envelope Consequences of virus infection in animal cells Classic lytic cycle Classic lysogenic cycle Transformation Conversion of a normal cell into a tumor cell Persistent infections Release of virions from host cell does not result in cell lysis Infected cell remains alive and continues to produce virus Latent infections Delay between infection by the virus and lytic events Cell fusion Two or more cells become one cell with many nuclei The Specific Example of HIV See video in notes Type Class VI notes Subviral Entities Defective Virusesvirus particles that are missing part or all of their genome Because of these deletions in their genome DIPs cannot sustain an infection by themselves Instead they depend on coinfection with a suitable helper virus The helper virus provides the gene functions that are absent from the DIPs Usually the small genomes of the DIPs are more efficiently replicated than the full length viral genome generating a very large number of noninfectious particles Viroids plant pathogens that consist of a short stretch of highly complementary circular ssRNA Prions infectious agent consisting of a protein in a misfolded form The correctly folded protein is often referred to as PrPC The misfolded protein is often referred to as PrPSC The misfolded protein PrPSC can effectively replicate by transforming the correctly folded protein PrPC into a misfolded protein 0 endogenous PrP interaction between PrP Spontaneous 3 generation of PrP and prp Conversion of mutant PrP to PrP39fH Inoculation of PrP H conversion of PrP to PrP accumulation of PrP Ex Mad cow disease accumulation of prions in the brain incurable Prion disease occurs by three distinct mechanisms Spontaneous generation of PrPSC Very rarely a normal PrP will randomly misfold to yield a PrPSC Conversion of a mutant Prp to PrpSC Some individuals carry an inherited mutation in Prp that makes it more likely to misfold Inoculation of PrpSC The misfolded PrpSC between animals or humans Lecture 22 Gene Regulation Constitutively expressed gene is expressed at the same level all the time Facultatively expressed gene is expressed only when needed The regulation of gene expression helps conserve energy and resources Which genes are expressed in a cell depends on its state and environment In metabolically active cells typically genes encoding subunits of RNAP and the ribosome are always being expressed Cell cycle genes like ftsZ are typically only expressed in the stage of the cell cycle that they are needed The different stages of growth require different genes In highly dormant states such as endospores almost none of the genes may be expressed In complex multicellular organisms with differentiated cells different cell types need to express different genes Two major levels of regulation in the cell One controls the amount of gene product Regulate level of transcription Regulate tRNA rRNA and mRNA degradation Regulate translation Regulate protein degradation Can take minutes to respond to a change in the environment One controls the activity of the product tRNA and rRNA modification Protein modification Can take seconds to respond to a change in the environment The allosteric enzyme End product allosteric effector Starting substrate 0 Active site Allosteric site NEnzymeA Intermediate Enzyme w Enzyme B l Substrate I INHIBITION ACTIVITY Substrate cannot Enzyme reaction lntermed39ate H 39 Fe dP bind enzyme proceeds 39 Inhlbltlon reaction inhibited Enzyme C I Intermediate III I Enzyme D l 1 End product w REGULATION OF TRANSCRIPTION DNA Binding Protein REGULATE TRANSCRIPTION FACTOR WW 39 WW T BINDING SITE The initiation of transcription is typically regulated by proteins called transcription regulators that bind to the dsDNA Most DNAbinding proteins interact with DNA in a sequencespecific manner The snecificitv is provided bv interactions Multiple outcomes after DNA binding are possible 1 DNAbinding protein may catalyze a specific reaction on the DNA molecule ie transcription by RNA polymerase 2 Negative regulation involving the binding event blocking transcription Positive regulation involving the binding event can activating transcription Many transcription factors exist as homodimers A homodimer is a quatinary structure that consists of two identical proteins Typically a homodimer recognizes inverted repeats in the DNA Each protein in the homodimer binds to one of the repeats Leucine zipper Leucine Helixturnhelix Zinc nger Helix Zipper Turn 2 3 b v Helix Zinc ions Fingers 439 w my quotI l39 Ilt ll llaI 4 A Mini 0 tllwlw Zinc Finger The above diagram illustrates the zinc finger DNAbinding domain The structure is stabilized by zinc ions In a DNAbinding protein with a zinc finger typically there are two to three zinc fingers HelixturnHelix The above diagram illustrates the helixturnhelix DNAbinding domain The first helix is the recognition helix The second helix is the stabilizing helix Many different DNAbinding proteins in bacteria contain helixturnhelix including the Lac and Trp repressors of E coli Leucine zipper The above diagram illustrates the leucine zipper DNAbinding domain Leucine residues are spaced every seven amino acids Does not interact directly with DNA Negative Control of Transcription The negative regulation of the transcription of an operon involves a transcriptional repressor that blocks transcription Negative regulation can involve induction or repression In the case of induction an operon that is normally not transcribed is transcribed In the case of repression an operon that is normally transcribed is no longer transcribed Since most operons are normally not transcribed induction is much more common than repression Transcriptional repressors often bind to a region called the operator downstream of the promoter to which RNAP binds and upstream of the operon The repressor sterically blocks transcription Effectors affect transcription indirectly by binding to transcriptional regulators Often the effector changes the conformation of the transcriptional regulator by binding to an allosteric site distinct from the DNAbinding site An effector that inactivates a transcriptional repressor is called an inducer An effector that activates a transcriptional repressor is called a corepressor Transcription proceeds Repressor Transcription blocked Repressor Positive Control of Transcription The positive regulation of the transcription of an operon involves a transcriptional activator that promotes transcription An activator often binds to a region called the activatorbinding site upstream of the promoter to which RNAP binds By themselves promoters of positively controlled operons only weakly bind RNAP The activator helps RNAP bind to the promoter either by interacting with RNAP or changing the local conformation of the DNA The activatorbinding site may be close to the promoter or several hundred base pairs away Transcriptional Regulatory Networks An operon can be regulated by multiple transcription factors including both repressors and activators Regulon A set of operons controlled by the same transcriptional In a cell there can be many overlapping regulons controlled by transcriptional activators and repressors Thus exquisite control can be achieved Two Component Regulatory Systems A twocomponent system TCS serves as a basic stimulusresponse coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions A TCS consists of a membranebound histidine kinase HK that senses a specific environmental stimulus and a cytoplasmic response regulator RR that mediates the cellular response TCSs are widely occurring in prokaryotes TCSs are also quite common in archaea Only a few TCSs have been identified in eukaryotes In E coli there are almost 50 TCSs Some environmental stimulus serves as the initial signal The stimulus can be a small molecule a peptide or even some larger biomolecule The stimulus binds to the HK and activates it The active HK autophosphorylates itself by transferring a phosphate group from ATP to one of its own His residues Then the active HK transfers the phosphate group to an ASP residue on the RR thereby activating it The RR is often a transcriptional regulator Regulation of Chemotaxis in E coli 0 Environmental O O Stimulus Histidine Kinase Response Regulator Differential gene expression Chemical gradients are sensed through multiple transmembrane receptors called methylaccepting chemotaxis proteins MCPs which vary in the molecules that they detect A MCP is bound to the proteins CheW and CheA with CheA being a kinase adds a phosphate group P03 to proteins In the absence of the attractant the kinase CheA is active CheA autophosphorylates to yield CheAP and then phosphorylates CheY to yield CheYP Interaction between CheYP and the agellum makes the agellum tend to rotate CW and hence the bacterium to tumble Another protein CheZ dephosphorylates CheYP to yield CheY The short halflife of CheYP means that the bacterium is very responsive to changes in the attractant concentration In the presence of the attractant the MCP undergoes a conformational change that inactivates the kinase CheA so CheA does not autophosphorylate and does not phosphorylates CheY When the agellum has no CheYP to interact with the agellum tends to rotate CCW so the bacterium tends to run Two other proteins CheR and CheB give the system a quotmemoryquot CheR is a methylase adds a methyl group CH3 to proteins CheB is a demethylase When the attractant is present MCP undergoes a conformational change The new conformation of MCP is a good substrate for CheR Therefore CheR adds methyl groups to the MCP When the attractant is absent and so the kinase CheA is active CheA phosphorylates CheB to yield CheBP and In the presence of the attractant CheR continually methylates the MCP and there is no CheBP to demethylate the MCP Therefore the MCP becomes increasingly methylated The more methylated the MCP the less sensitive it is to the attractant Therefore even more attractant is needed to produce a conformational change in the MCP inactivate the kinase CheA stop the formation of CheYP and make the bacterium run If the concentration of the attractant is high but constant the bacterium will start tumbling a lot again CheBP removes methyl groups from the MCP The more methylated the MCP the less sensitive it is to attractant In the absence of the attractant the MCP is in its normal conformation so CheR can no longer methylate it and the kinase CheA is active so CheBP is being produced and can demethylate the MCP Therefore the system returns to its initial state Lesson 23 Quorum Sensing Overview of Mechanism Quorum sensing is a mechanism by which bacteria assess their density It ensures that there are a sufficient number of cells present before initiating a response that requires a certain cell density to have an effect Each species of bacterium produces a specific autoinducer molecule There are several different classes of autoinducers Acyl homoserine lactone AHL was the first autoinducer to be identified At low cell densities the autoinducer just diffuses away At high cell densities the autoinducer accumulates The autoinducer usually regulates a transcriptional regulator and this regulator in turn regulates the expression of various genes Example of Aliivibrio scheri The Hawaiian bobtail squid lives in a symbiotic relationship with A fischeri The A fischeri inhabit a special light organ in the squid39s mantle The bacteria are fed a sugar and amino acid solution by the squid In return the bacteria hide the squid39s silhouette when viewed from below by matching the amount of light hitting the top of the mantle The luminance of A fischeri is governed by the Lux quorum sensing system The key genes are arranged as shown The diveregently transcribed operons luxR and quICDABE are separated by an intergenic region called the M box LuxR is a transcriptional regulator LuxI synthesizes an autoinducer 3oxoc6HSL LuxCDABE are the enzymes that generate light by catalyzing the reaction FMNH202RCHO gt FMN R COOH H20 Light At low cell densities The gene luxR is expressed at a high level But the resulting transcriptional regulator LuxR is inactive The operon quICDABE is expressed only at a low level The resulting small amount of LuxI synthesizes only a small amount of autoinducer 3oxoc6HSL The autoinducer diffuses away into the surrounding medium l I gt fwd luxbox de C D A B E W ccll density l 39 30xoC6HSL O quot l l It ll V 0 At high cell densities The autoinducer begins to accumulate It binds to and activates the transcriptional regulator LuxR LuxR binds to the M box and represses the expression of luxR and activates the expression of IuXICDABE The resulting LuxCDABE generate light quR qubox nd hit h cell density 0 O O 3 oxoCBHSL O o k 0 0390 39 0 o 00 Example of Staphylococcus aureus S aureus is a grampositive bacterium that is frequently found in the human respiratory tract and on the skin It especially likes to live in the nose It is a common cause of skin infections eg boils respiratory disease eg sinusitis and food poisoning It also sometimes can cause serious lifethreatening disease It is not always pathogenic About 30 of people are colonized with S aureus The progression of an S aureus infection is illustrated Bacteria reversibly attach to a solid support This can be a cut in the skin The bacteria aggregate to form a matrix At low cell densities there are enough nutrients so the bacteria rapidly replicate At high cell densities nutrients will soon become limiting so the bacteria express virulence factors that allow them to disperse and invade This can involve entering the bloodstream and migrating to other sites in the body to found new infections The expression of virulence genes at high densities is governed by the Agr quorum sensing system The key genes are arranged as shown The operon agrBDCA is transcribed from the promoter P2 The operon RNAIII is transcribed from the promoter P3 AIP Heterologous AIP AgrB J 9quot A coo4 f fRNAIII AgrD p2 P3 1 mm ang D agrB URNAIII ltxotoxins l 3 Surface proteins Variable region AgrD is the cytosolic preform of the autoinducer It needs to be processed to yield the final autoinducer AgrB is a transmembrane protein that processes AgrD to yield extracellular autoinducer AIP Ang is a transmembrane kinase that senses the autoinducer AgrA is the corresponding response regulator that regulates transcription RNAIII is a regulatory RNA that upregulates the expression of virulence genes At low cell densities The operon agrBDCA is expressed only at a low level The resulting small amount of autoinducer AIP just diffuses away The gene RNAIII is not expressed at all There is no RNAIII to upregulate the expression of virulence genes At high cell densities The autoinducer AIP accumulates It binds to and activates the kinase Ang Ang phosphorylates and thereby activates the response regulator AgrA AgrA increases the transcription from the promoters P2 and P3 This leads to the expression of RNAIII RNAIII upregulates the virulence genes Stringent Response Stationary Phase At high cell densities The autoinducer AIP accumulates It binds to and activates the kinase Ang Ang phosphorylates and thereby activates the response regulator AgrA AgrA increases the transcription from the promoters P2 and P3 RNAIII upregulates the virulence genes Review of Translation Translation involves the ribosome attached to a mRNA transcript The ribosome has three sites E P and A At first the P site is at the start codon AUG coding for Met Translation elongation begins when a tRNA carrying the amino acid Met attaches to the start codon on the mRNA at the P site The next charged tRNA with its amino acid attaches to the next codon at the A site Here the amino acid is Thr The ribosome catalyzes the formation of a peptide bond between the Met at the P site and the next amino acid at the A site The tRNA at the P site is now uncharged The tRNA at the A site is bound to the polypeptide Here the polypeptide chain is Met Thr The ribosome moves in the 339 direction down the mRNA by one codon shifting the tRNA with the polypeptide chain to the P site and the uncharged tRNA to the E site The A site is now open The uncharged tRNA at the E site is ejected The process repeats over and over again until a stop codon is encountered The stringent response in E coli When there are no amino acids the tRNAs remain uncharged With no charged tRNAs the ribosome stalls The stalling of the ribosome is sensed by a protein RelA that is associated with the ribosome RelA catalyzes the reaction ATP GDP gt AMP ppGpp The molecule ppGpp is called an alarmone as it is a sign that the cell is under stress The alarmone ppGpp binds to and modulates RNAP The modulated RNAP transcribes different genes Regulation of sporulation in B subtilis Earlier we learned about sporulation in B subtilis Sporulation is governed by a phosphotransfer relay system whose mechanism resembles that of a two component system but is considerably more complex You do not need to memorize the phosphotransfer relay system Just be aware that one protein can phosphorylate and thereby activate another then that protein can phosphorylate and thereby activate yet another and so on Lesson 24 Genetic Mutation Mutation Heritable change in DNA sequence that can lead to a change in phenotype observable properties of an organism Mutant A strain of any cell or virus differing from parental strain in genotype nucleotide sequence of genome Wildtype strain Does not carry mutations by definition Selectable mutations Those that give the mutant a growth advantage under certain conditions Nonselectable mutations Those that usually have neither a growth advantage nor disadvantage over the parent Induced mutations Those made environmentally or deliberately Can result from exposure to natural radiation or oxygen radicals Spontaneous mutations Those that occur without external intervention Point mutations Mutations that change only one base pair Can lead to single amino acid change in a protein an incomplete protein or no change at all Silent mutation Does not affect amino acid sequence Missense mutation Amino acid changed polypeptide altered Nonsense mutation Codon becomes stop codon polypeptide is incomplete Frameshift mutations Deletions or insertions that result in a shift in the reading frame Often result in complete loss of gene function when within a gene Reversion Alteration in DNA that reverses the effects of a prior mutation Revertant Strain in which original phenotype is restored Two types 0 Samesite revertant mutation is at the same site as original mutation Secondsite revertant mutation is at a different site in the DNA suppressor mutation that compensates for the effect of the original mutation Selection vs Screening 0 Under selection individuals with advantages or quotadaptivequot traits tend to be more successful than their peers reproductively meaning they contribute more offspring to the succeeding generation than others do When these traits have a genetic basis selection can increase the prevalence of those traits because offspring will inherit those traits from their parents When selection is intense and persistent adaptive traits become universal to the population or species which may then be said to have evolved abrupt selection ex plating bacteria with antibiotics plating one without 0 Gradual selection wild type and mutant in one culture N wild t Z N wild0 hr X 2Hng Nmutamo Nm 0 hr x 2tgmutam utant Nmutant Nmutant Nwild Nmutant 1 Nmutant tNwild f Mandi Screening 0 In the case of selection the mutants being isolated have a growth advantage so the mutants will come to dominate the population over time 0 Screening involves the isolation of mutants that do not necessarily have a growth advantage and so can be much more tedious Mutation Rates 0 For most microorganisms errors in DNA replication occur at a frequency of 109 tolO10 per base 0 DNA viruses have error rates 100 1000X greater 0 The mutation rate in RNA genomes is 1000fold higher than in DNA genomes Some RNA polymerases have proofreading capabilities However RNA repair mechanisms similar to DNA repair mechanisms do not exist 0 Perfect fidelity in organisms is counterproductive because it prevents evolution 0 The mutation rate of an organism is subject to change Mutants can be isolated that are hyperaccurate or have increased mutation rates 0 Deinococcus radiodurans is 20 200 times more resistant to radiation than E coli Mutagenesis Mutagens Chemical radiation or biological agents that increase mutation rates Several classes of chemical mutagens exist Nucleotide base analogs Resemble nucleotides Chemical mutagens that induce chemical modifications For example alkylating agents like nitrosoguanidine Chemical mutagens that cause frameshift mutations For example intercalating agents like acridines Two main categories of mutagenic electromagnetic radiation Nonionizing ie UV radiation Purines and pyrimidines strongly absorb UV Pyrimidine dimer is one effect of UV radiation Ionizing ie Xrays cosmic rays and gamma rays Ionize water and produce free radicals Free radicals damage macromolecules in the cell The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause cancer More formally it is a biological assay to assess the mutagenic potential of chemical compounds A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen because cancer is often linked to mutation The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound because standard carcinogen assays on mice and rats are timeconsuming taking two to three years to complete and expensive The Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis These strains are quotauxotrophicquot mutants ie they require histidine for growth The method tests the capability of the tested substance in creating mutations that result in a reversion back to a quotprototrophicquot state so that the cells can grow on a histidinefree medium DNA Repait Mechanisms 0 Three Types of DNA Repair Systems Direct reversal mutated base is still recognizable and can be repaired without referring to other strand Repair of single strand damage damaged DNA is removed and repaired using opposite strand as template Repair of double strand damage a break in the DNA 0 Requires more errorprone repair mechanisms 0 When DNA damage is large scale the cell may use a different type of repair system ie damage interferes with DNA replication Mechanism called the SOS regulatory system 0 This system is more error prone Allows replication to proceed and cell to replicate but errors are more likely Translesion synthesis allows DNA to be synthesized with no template Lesson 25 Gene transfer between Bacteria Genetic Recombination Genetic recombination is the process by which two DNA molecules exchange genetic information resulting in the production of a new combination of alleles Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA Donor DNA Endonuclease nicks DNA NICE Binding of 883 protein 333 proteln 00 oo Recipient Strand RecA invasion protein 00 r O Development of crossstrand exchange vquot aunt r quot 4 III 39 I Resolution quotResolution at sites 319122 Patches Splices F tanking sequence Eiaulerzul 13 The transgene can be integrated into a precise locus one only has to flank the transgene with sequences similar to that of the insertion site desired 39ar sger e Plasmid p asmgd Recombination NA event tt Ullal DNA I quotar39sge39ie 9 Insertion site Insertion site Mediating two recombination events between the flanking sequences 0n the plasmid and their homologue in the bacterial DNA the transgene has been inserted into the desired locus Transformation Transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous DNA For transformation to happen the must be in a state of competence In some species competence naturally occurs under common conditions such as starvation In other species competence can be induced by artificial means such as electric shock heatshock and chemical induction Transfection is the transformation of a cell with DNA extracted from a Virus Transduction Transduction is the process by which DNA is transferred from one cell to another by a Virus Conjugation Conjugation is the transfer of genetic material plasmid between cells by direct cell tocell contact or by a bridgelike connection between two cells Circular DNA molecule 100 kbp Contains genes that regulate DNA replication Contains several transposable elements that allow the plasmid to integrate into the host chromosome Contains tra genes that encode transfer functions Presence of the F plasmid results in alterations in cell properties Ability to synthesize F pilus Mobilization of DNA for transfer to another cell Alteration of surface receptors so that cell can no longer act as a recipient in conjugation Hfr Conjugation A highfrequency recombination cell Hfr cell also called a Hfr strain is a cell with a conjugative plasmid often the Ffactor integrated into its genomic DNA Unlike a normal F cell Hfr strains will upon conjugation with a F cell attempt to transfer their entire DNA F plasmid is an episome can integrate into host chromosome Cells possessing a nonintegrated F plasmid are called F Cells possessing an integrated F plasmid are called quot high frequency of recombination 0 High rates of genetic recombination between genes on the donor chromosome and those of the recipient Insertion sequences mobile elements are present in both the F plasmid and E coli chromosome Facilitate homologous recombination Plasmid is now part of chromosome Chromosomal genes transferred with plasmid Recipient cell does not become Hfr because only a portion of the integrated F plasmid is transferred by the donor Archaea Bacteria Archaea contain single circular chromosome Genetic manipulation of Archaea lags behind Bacteria Archaea need to be grown in extreme conditions Most antibiotics do not affect Archaea No single species is a model organism for Archaea Examples of transformation transduction and conjugation exist Transformation works reasonably well in Archaea Transposable Elements Transposable elements are discrete segments of DNA that move as a unit from one location to another within other DNA molecules Transposable elements can be found in all three domains of life Move by a process called transposition Frequency of transposition is 1 in 1000 to 1 in 10000000 per generation First observed by Barbara McClintock Two main types of transposable elements in bacteria are transposons and insertion sequences Both carry genes encoding transposase Both have inverted repeats at their ends Insertion sequences are the simplest transposable element 1000 nucleotides long Inverted repeats are 10 50 base pairs Only gene is for the transposase Found in plasmids and chromosomes of Bacteria and Archaea Found in some bacteriophages 0 Transposons are larger than insertion sequences Transposase moves any DNA between inverted repeats May include antibiotic resistance genes 0 Examples are Tn5 and Tn10 Insertion of a transposable element generates a duplicate target sequence 0 Using transposons to make mutants Transposons with antibiotic resistance are used Transposon is on a plasmid that cannot be replicated in the cell Cells capable of growing on selective medium likely acquired transposon Most insertions will be in genes that encode proteins Next step screen for loss of function to determine insertion site Target DNA sequence ABCD 13 E I13 IE Transposable element Insertion ABCIDIE BABCD B LDuplicated target sequence J
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