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55-140 (56)
Habetler (53)
Lecture

Molecules and Cells

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Department
Biological Sciences
Course
55-140
Professor
Habetler
Semester
Fall

Description
Molecules and Cells Molecular Biology and Genetics Lecture 1: DNA, RNA Synthesis: DNA Recombination See hand written notes 1 Lecture 2: Mutation and DNA Repair Why Mutation is an Important Phenomenon Best seen with organisms that replicate quickly-bacteria/viruses Antibiotic/antiviral resistance-changes in the characteristics of disease causing organisms Ex: bacteria, AIDS (AZT)-selects for resistant organisms CAD-genetic ties to MI’s- 1/500 humans have a mutation in the gene that codes for low density lipoprotein (takes cholest out of blood- high serum cholest) Predisposition to CA Production of vaccines ex: polio vaccine-positive implication-attenuated virus-enough mutations that it doesn’t cause dz Chemistry of Mutations Example: bacteria and their ability to grow on certain mediums wt- can synthesize all essential compounds, can grow on any compound X mutan only grow in things distal to the block, no way to convert to final Cell will try to make more of what it needs to get to the next step Sometimes defective in the proteins that decide whether to start a new round or not, takes a while for the kinetics to level off-just not starting new ones How to find mutants Examine them one by one, such as in the medical system-medical literature Looking at simple organisms-use a selection-protocol that eliminates (kills) all non-mutants, ex: want to get all bacteria resistant to certain abx, apply the abx When confers a disadvantage-grow bacteria in PCN-cells that aren’t dividing are very resistant to PCN-those who are dividing will be killed Ex: Proline oxotroph-can make praline autotroph-can’t Find it by taking e coli and grow them in the absence of proline-the mutants who can’t make it are not growing -those who can killed when add PCN b/c dividing What you can learn from genetic analysis Plaque-clonal growth from single virus particle Could ask how many genes reside in that collection of mutations Complementation Test Each of the two mutants being tested are used to infect the same cell simultaneously If they are each defective in one- then they would be successful in getting new progeny-would conclude different genes compliment If defective on the same gene, still no protein produced-fail to compliment No exchange of genetic material 2 Reverts True Revert-reverts back at the same site Second Site Revert-mutation somewhere else corrects the defect Intragenic suppression-same gene Ex: protein can’t fold right, bump in side chain-change another aa somewhere else to prod a space that accommodates this side chain Protein dimerizes-make a bump on one, and another will make a groove Extragenic suppression-different gene Ex: pathway controlling cell growth-activating a parallel pathway Powerful way of defining fxn in the cell What Mutations do you see? It’s NOT random Often cluster in same area Mechanisms Spontaneous Slippage and loop out during DNA replication Short sequences that share identity ex: GTGTTGAA G-C to A-T base changes High frequency occurs in places where cytosine is methylated Enzymes patrol DNA for uracil and assume base opposite is correct-if C is methylated (controlling strxr of chromatin) and then de-aminated you now have thymine opposite of guanine C to T mutations (in us too!) Induced Treating with mutagens-proteins that maintain fidelity of the genetic material are damaged and the rate of mutations are increased-irrelevant, plays no rules Can also have chemicals directly damage the DNA- can study this on simple organisms b/c have same DNA as us and can also look at chemistry of the damage Test by Ames Test-create a set of mutants that can’t make Histidine- then select for mutants that can revert-get a reversion rate-then find out exactly what the mutation is Ex: Aflotoxin- some strains not affected, some very sensitive- can study reverts and see what the mutations are Many carcinogenic compounds become carcinogenic in the liver Many cause an ambiguity in the base pairing-creating by chemical modification an ambiguity or even switch Planar aromatic compounds promote single base slippage-stabilize the slippage transition state 3 DNA Level Changes that affect coding region Ex: mRNA Missense mutation-one aa traded for another (many can survive this) Nonsense mutation-codes for a stop codon Frameshift mutation-put in or take out 1 or 2 extra nucleotides-bad news- affects downstream Silent mutation-degenerate code, not seen Suppressor Mutations Translational suppressors-mutated tRNA-different anticodon-puts in wrong aas Most are encoded by multiple tRNAs and not likely this would happen in all of them Repair of Damage Mutation on of the few single events in biology that can bring an organism to its knees- single event in a single cell can do it in, must find a way to fix the damage DNA double stranded-so have another copy to recover the information Have enzymes to fix, controlled by the damage itself Ex: take ds DNA virus and irradiate with UV light-get a response curve-dose v. light-measure survival with plaques-do the same with E. coli that have been irradiated first-get a different survival curve-larger number survive-b/c repair enzymes had been induced and were waiting Repair Mechanisms for… UV Exposure- thymine dimer as a result of UV radiation-photoreactivation enzyme recognizes these and uses visual light to do a photochemical reversal Any chemical lesion that distorts DNA (not just UV)-enzyme recognizes-Cut upstream and downstream, gap repaired by DNA polymerase using the other strand-extremely important in us-can test with complementation by fusing nuclei from two patients and scoring resistance to see if defect in same gene Alkyl groups attaching-methyl acceptor protein with free cysteine picks up the methyl group-suicidal for protein-but it’s there to sacrifice itself for the greater good of the cell-can also accept from the phosphate backbone with another cysteine-also a transcriptional activator for its own gene to make more of itself Mismatch Repair Assume that the template is the right one-wants to correct with the template E coli does this by epigenetic modification-methylation Enzymes patrol cell looking for hemimethylation in mismatches-slides down strand and knows which is which Problems with this lead to CA-frequency of errors increases greatly-this process is central to keeping us from becoming huge blobs of CA cells 4 Lecture 3: Genetic Code and Protein Synthesis See attached notes for first two pages Basic Steps of Translation 1. Initiation load ribosomes on mRNA and find the right AUG start codon Problem of how the ribosome and the initiator tRNA can find it Similarities and differences Bacteria Eukaryotes Both • Shine-Delgarno • Dramatic • Have a start and stop codon • Polycistronic modifications • AUG (and sometimes GUG • DNA each a different or UUG) code for initiator protein methionine tRNA • Termination code-UAG, UGA or UAA Bacterial Initiation Shine-Delgarno is engaged and three IFs bring f-Met-tRNA fetto the P site Shine-Delgarno • A polypurine tract approx 6 to 8 bases upstream of AUG start site o Loosely complementary to the 16S rRNA (polypurimidine recognizes polypurine) component of the small ribosomal subunit-allows for tethering of mRNA o Polypurine tract correlates to how well those genes are expressed A GTPase is involved in the ribosomal subunit joining the first step Eukaryote Initiation • Similar process, identification of the initial AUG is more complicated and dependent on cap and the polyA tail • Complex of proteins assembles at the cap strxr, they interact with the polyA tail to form a closed loop complex ready for translation • Initiation factor (eIF2) binds to a ternary complex with Met-tRNA and GTP-the AUG initiator codon located by scanning o Scan for AUG in 5’ to 3’ direction, uses the first one it finds 2. Elongation extend the growing chain by one aa 3 Basic Steps 1. A-site loading mediated by EF-Tu (EF-1) o Discrimination of codon-anticodon interaction o Aminoglycoside antibiotics (neomycin, paromycin, gentamycin, etc) bind to the small subunit in the decoding center-increased rates of miscoding  They tend to bind more tightly to bacterial than eukaryotic ribosomes (why it works) 5  NMR studies have shown where they bind first  Helps us to understand the common mechs for resistance 2. Peptide bond forms • Chemical joining of two aas • Simple chemical reaction with a good Nu attacking an activated, labile aminoacyl ester bond and the transfer of the growing peptide chain to the aminoacyl (A) tRNA • This step is targeted by abx like erythromycin, azithromycin- all must primarily bond to the RNA-rich active site (transition state analogue surrounded by nothing but RNA) o Diverse abx bind in the same vicinity of ribosome strxr o All target peptide bond formation directly or the extension of the chain o Mutation of rRNA gene, r-proteins o Acquisition of rRNA methylase 3. Translocation of the mRNA:tRNA complex-catalyzed by EF-G (EF-2) Moves the complex 3 codons forward Must empty A site for next cycle to begin EF-G hydrolyses GTP and promotes translocation How Resistance Comes About • Nucleotide changes in the rRNA • Direct modification of critical positions of the rRNA or the abx • Transport system • Substitution of base pairs 3. Termination recognizes the termination codon and release the peptide chain • Release factors (RF1 and RF2) recognize (NOT tRNA) the set of stop codons and stimulate the hydrolysis of the growing peptide chain • GTP hydrolysis is again involved 4. Ribosome Recycling release the ribosome from the message for subsequent rounds of translation-RF-3, EF-G, IF3 and RRF Protein Factors Involved in all steps of protein synthesis Bacterial Eukaryotic 6 Initiation IF1 eIF1A IF2 eIF5B IF3 eIF1 (fidelity of AUG) eIF2(tRNAmet) eIF4E (cap) eIF4G (scaffold) eIF4A (helicase) eIF4B (RNA Binding) eIF4B (RNA Binding) eIF3 (unknown, large) eIF5 (GAP for eIF2) Elongation EF-Tu eEF-1a EF-G eEF2 Termination RF1 eRF1 RF3 eRF3 Recycling RRF No known homologue in eukaryotes Ribosomes Bacterial Eukaryotic Small Subunit 30S 40S Large Subunit 50S 60S Total 70S 80S Many clinically relevant abx target the ribosome Targets are primarily RNA High conservation makes targeting the ribosome a challenge Rx needs to be able to find things that are different in the target Ex) linezolid- new last-line drug, synthetic compound Translation Regulation • Most common during initiation o Many signaling pathways impinge on translation and limit the rate of initiation  Ex) insulin, aa starvation • Important for understanding development and memory, and because viruses often hijack this process 7 Lecture 4: Molecular Genetics Methods I Restriction Enzymes Cut across both strands of the DNA molecule at a specific and small (4-8bp) recognition sequence Also function to destroy infecting bacteria Host DNA is protected by methylation Many cut to give sticky ends-can be efficiently rejoined with DNA ligase Plasmids Convenient vectors for cloning DNA Typically several kilobases with an origin of DNA replication, abx resistance gene and a region not essential for propagation where a piece of foreign DNA can be inserted Can produce some protein from the cloned DNA Small proteins that fold easily on their own Ex) growth hormone production-recombinant source Libraries Collection of DNA segments that are inserted into a cloning vector , typically a plasmid or virus Genomic DNA Libraries cDNA Libraries Complimentary DNA libraries DNA copies of mRNAs in cells Useful b/c only a small percent code for proteins Denaturation and Renaturation of DNA or RNA = “hybridization” Direct consequence of base pairing specificity of double stranded DNA or DNA-RNA hybrid After the hybridization rxn is complete, the unbound probe is washed away and the location and the amount of bound probe determined Denatured by heating or high pH Applications Southern (DNA) blots, Northern (RNA) blots, in situ hybridization Detecting DNA (or Proteins) Radioisotopes- disadvantages finite life of isotopes, requirement of nuclear reactor, small health risks Enzymatic detection-DNA derivatized so that it can bind to the protein that is readily detectable Fluorescence Restriction Enzyme Cleavage Maps & Southern Blotting Agarose gel electrophoresis 8 Southern blotting: paper towels, nitrocellulose, agarose gel, buffer soaked filter paper Can do whole genome Southern blotting Synthetic DNA Now routine to chemically synthesize DNA up to 100 bp in length Done on a solid phase with the growing nucleotide chain anchored to a resin and the nucleotide to be added to the chain in solution Growing chain treated after every reaction to remove the protecting group Applications: synthetic genes, sequencing and PCR primers, etc DNA Sequencing by the Sanger Method 1. Hybridize primer and template 2. Add DNA polymerase, dATP, dTTP, dGTP, dCTP with a radioactive phosphate in the first position and a little bit of ddATP (following the incorporation of this, the chain can’t elongate) 3. Do three more reactions with dTTP, dGTP, dCTP 4. Denature template from primer by heating and then run the DNA on a polyacrylamide gel to separate chains by size Polymerase Chain Reaction (PCR) Similar to cloning DNA by propagation of recombinant molecules in bacteria Gives an amplification of a particular sequence-each amplification takes only minutes Applications: forensic amplifications, site-directed mutagenesis, reverse transcriptase PCR, assembling complicated arrangements of DNA sequences DNA chip Hybridization Miniaturized array of target DNA sequences immobilized on a solid surface Applications: mutation detection, chromatin immunoprecipitation 9 Lecture 5: Molecular Genetics Methods II Detecting and Purifying Proteins Western Blotting Using antibodies to detect proteins separated by gel electrophoresis 1. separate denatured proteins by molecular weight using SDS/PAGE gel electrophoresis 2. Filter with proteins 3. Filter with antibodies, localized to one band only Tagging Proteins Popular sequences to add: 1. other proteins that an easily be purified because of their affinity for simple molecules 2. 6-8 histidines confers binding to a solid support with partially chelated Nickel ions 3. The binding sites of available monoclonal antibodies Yeast two hybrid screening Purely genetic approach-identifies which other proteins bind to a particular protein of interest Activates transcription of a modified his3 gene that is under Gal 4 control and allows the yeast cell to grow on plates lacking histidine Tissue Culture Mammalian Tissue Cx Dispersed cell and slice cx Growth medium: buffer, salts, amino acids vitamins, calf serum, phenol red Need large surface to volume ratio to get enough diffusion Applications: propagating and studying animal viruses, determining chromosome constitution, identifying chemical basis of genetic defects, study cell function, transfection (intro of DNA into cultured cells), and factories to produce useful proteins Production of Monoclonal Antibodies A wedding of immunology and tissue cx Immunize animal, isolate spleen cells, select hybrid cells in HAT medium, wait two weeks for cells to grow, test medium from each well for the presence of antibodies directed against the antigen Transgenisis and Gene Targeting in Mice Producing transgenic mice Mate male and female mice, harvest fertilized egg, microinject DNA into pronucleus, DNA integrates randomly, don’t know where it goes or how many copies, implant eggs in foster mother, and analyze pups by PCR of tail DNA 10 Homologous Recombination in Embryonic Stem Cells Allows targeted mutation of any gene in the mouse Introduce DNA segment into embryonic stem cells and select for homologous integration, want two crossover events, G418 selects for neoR ganciclovir selects for TK-Inject ES cells with correct targeting event into blastocysts and implant blastocysts into foster mother. Pups will be chimeras (as seen by coat color) Recent Advances in Genetic Manipulation of Mice Site specific DNA recombination: Cre and Flp are proteins from a bacterial virus and yeast which catalyze site specific recombination with DNA targets that are very large Gene Therapy Somatic v. germline therapy, who it affects and ethical considerations Random events occur with high frequency and are very hard to avoid Genetically Engineered Vaccines Much more efficient than past methods Stem Cell Therapy Grafting in cells that can appropriately differentiate and integrate into the body 11 Lecture 6: Bacterial Cells Prokaryotes Differ from Eukaryotes No membrane bound nucleus Usually have a single circular chromosome Genes interrupted by introns Ribosomes 70s instead of 80s Cell walls contain muramic acid (except Archaea) No mitochondria or membrane bound organelles No cytoskeleton or endocytic vesicles Movement by flagellar rotations Two Groups Eubacteria true bacteria, human pathogens and cyanobacteria (blue-green algae) Archaea methanogens, thermophiles, halophiles-ancient line of life Cell walls lack muramic acid Membrane lipids contain glyceryl ethers (rather than esters) Membrane lipids contain polyisoprenoind chains (rather than alkyl) Carl Woese discovered that all living things are derived from Eukaryotes, Eubacteria and Archaea Classification of Prokaryotes Gram Stain Uses crystal violet and then fixed with iodine Basis for differential staining is the thickness of the cell wall Positive-purple Negative-red Shapes single cocci cocci in a tetrad vibrio cocci in pairs coccobacilli spirillium cocci in chains bacilli with rounded ends spirochete cocci in clusters bacilli with square ends Molecular Taxonomy Common ancestry leaves residual similarity that is greater for related than unrelated individuals Creation of phylogenetic trees More recently, molecular bio, particularly 16s rRNA sequences have been used to determine relationships between different organisms 12 The Prokaryotic Cell Cell division by fission, no spindle apparatus, bacterial cell wall made of rigid peptidoglycan and contains muramic acid Organelles • No true organelles, certain bacteria have gas vesicles bounded by a protein layer • Carboxysomes are the site of carbon dioxide fixation in autotrophic organisms • Photosynthetic organisms may have chlorobium vesicles Granules • Non-nitrogenous organic reserve molecules, stored as starch or glycogen or poly-beta-hydroxybutate Nucleoid • Single, circular DNA molecule, no membrane Mesosomes • Convoluted invaginations of the cytoplasmic membrane • Septal mesosomes act in cross wall septum formation and nuclear segregation • Lateral mesosomes the site of active electron transport systems Cytoplasm • Contains soluble enzymes, ribosomes, mRNA, tRNA, storage granules • Bound by cytoplasmic membrane • mRNA synthesis and translation take place simultaneously Cell envelope • cytoplasmic membrane and cell wall • regulation of permeability and transport • electron transport and oxidative phosphorylation • site of enzymes for DNA and cell wall synthesis and membrane lipid synthesis Flagellum • swimming apparatus Pili • thin proteinaceous tubes projecting from bacterial surface • F+ pili are organs for conjugation and DNA transfer Periplasmic Space • Periplasmic proteins reside in the space between the inner and outer membrane Glycocalyx or capsule • Slimy layer of polysaccharides polymerized at the outer surface 13 • Sometimes consists of polypeptides Surface Structures Functional role of the capsule Attachment, protection, resistance to drying, reservoir for certain nutrients Flagella, motility and chemotaxis Flagellar filament is constructed from a single protein arranged in helical symmetry around a hollow core; rings in the basal body rotate relative to each other, energy obtained from protein motive force Different from flagella in eukaryotic cells Cell Envelope Gram - Gram + Have an outer membrane, and often have No outer membrane, Thicker than gram – periplasmic space Have polysaccharides covalently linked to Helical lipoproteins (Braun’s) covalently pepetidoglycan teichoic acids and lipoteichoic linked to the peptidoglycan anchor to the OM. acids, which penetrate the peptidoglycan layer No polysaccharides are bound to the from the cytoplasmic membrane peptidoglycan of gram – Teichoic acids found in two types: membrane Lipopolysaccharides are found in the outer type (polyglycerol phosphate polymer) and cell leaflet of OM, transmembrane porins occur wall type (attached to amino sugar chains, only in the OM polyglycerol phosphate or polyribitol Nutrients acquired through OM via porins phosphate) O-Antigen Lipoteichoic Acid Core Lipid A OM a phospholipid bilayer and is more permeable to charged ions than is the cytoplasmic membrane 14 Peptidoglycan Rigid layer-backbone composed of alternating polymer 2 sugars, N-acetyl glucosamine and N-acetyl muramic acid (NAM only in eubacteria) Attached to NAM side chain are 4 aa (tetrapeptide) Gram + third aa = lysine, Gram - =meso-diaminopimelic acid In both side chain lysines involved in cross-linking peptidoglycan 50% of total cell wt in Gram + and 1-2% in Gram – How Antibiotics Work PCN 1 Substrate for peptidoglycan linking is a pentamer with two D-ala groups at the 4 and 5 th positions-cross-linking enzyme recognizes D-ala D-ala 2 step rxn 1: nucleophile of the active site attacks D-ala D-ala amide bond acyl intermediate 2: Acyl intermediate attacked by an amine of DAP (Gram -) or amine of pentaglycine (Gram +) releases the acyl enzyme and replaces it with a peptide bond PCN works because its Beta lactam ring is a structural mimic between ala4 and ala5-acts as a competitive inhibitor of the transpeptidase as it attacks in Step 1 Permanently inactivates-“suicide inhibitor” Cidal Drugs Ex: PCN Turbidity and viable counts fall after administration of the drug Acts only on growing cells Static Drugs Ex: Chlorampheicol 15 Inhibits further growth, but does not kill the cells, which will grow well once the drug is removed Antibiotic Resistance Can result from cleavage or other modification of the drug Can be a change in the drug target, a pump that pumps the drug out of the cell, encoding a beta lactamase (what happens with PCN) Vancomycin and the Horizontal Transfer of Resistance Structurally unrelated to PCN-but targets same process Resistance is coded by a multigene operon-vanco can’t bind to the side chain, but its still a substrate for transpeptidase-can still be cross linked, but reduces binding to vanco by 1000 fold Important Genes in the van operon vanH A reductase that makes D-lactate from normal metabolites vanA D-ala D-lac ligase vanX D-alal D-ala dipeptidase Transfer of Resistance Transposons and plasmids The van containing plasmid can be transferred in the lab via sexual conjugation to enterococci and streptococci 16 Lecture 7: Bacterial Gene Transfer All comes down to DNA recombination Bacterial Viruses Viruses that infect bacteria (not people) Discovered in the early 1900’s-realized that it was an organism that could kill bacteria, leaving people unharmed-have not proven particularly useful in combating human infection Ex: (no details) Lambda-attaches and injects its DNA into the cell, two cycles so it doesn’t just kill all the hosts-not strictly a parasite-provides resistance to other viruses and other lambdas Lytic Cycle-explodes cell Lysogenic cycle-integrates into cell’s genome or is carried passively-host doesn’t seem to mind-silent genetically Gene Transfer Reassortment of genes within a population of organisms Occurs at meiosis in higher organisms Bacteria multiply by fission and there is no obligatory coupling-no bacteria actually fuse cells Transduction Transfer by a virus or virus-like particle Moving host genes around advantageous-recombination Transducing phage can: 1. integrate via homologous recombination via the transduced bact seqs 2. integrate by homologous recombination with an already integrated phage genome 3. persist as an extrachromosomal circle, if unable to crossover=abortive transduction 4. rarely, it can integrate into the chromosome by site-specific integration Ex: Cholera-dance between vibrio, bacteria and bacterial virus Transformation Direct uptake of free DNA Typically inefficient-usually less than 1% of cells transformed Ex: Demonstrated by the Griffith experiment 17 Some bacteria have a DNA uptake system that only takes its own DNA Ex: Model for transformation of streptococcus Conjugation Transfer by cell-to-cell contact Unidirectional transfer mediated by a plasmid Plasmid called an F-factor mediates Want to max their spread and survival regulate the expression of their transfer functions Tra operon-encodes the transfer functions, active following entry into recipient cells-cell becomes competent to reproduce Fin-fertility inhibition-inactivator of pilus receptors F+ cells-have the F-factor, F-pili F- cells-have receptors for pili F+ x F- mating Most common mating event, transfer of F plasmid only-no chromosomal DNA 1. site specific nick on the end of one strand of the F plasmid (endonuclease) 2. DNA replication from this site displaces a single stranded tail that is passes into the recipient cell where the complementary strand is synthesized 3. When the entire plasmid has been replicated, the result is two F+ cells Hfr x F- mating Occurs when the F plasmid integrates into the host chromosome F Factor integration via recombination between one of the many transposable elements in the F-factor Recipient remains F- Some genes are transferred more efficiently than others Chromosomal genes close to the site of F-plasmid integration and the side towards which ori-T initiated DNA replication are the most efficient F’ x F- mating F factor integration is reversible, and Hfr can revert to an F+ and an F+ can become and Hfr F’ result of a rare, F factor excision with adjacent cellular sequences excised with it 18 F’ formation is mediated b homologous recombination between transposable elements Analogous to transfer by lambda Upon entering recipient, F’ can: 1. replicate autonomously 2. exchange genetic material with the recipient chromosome by double recombination 3. recombination Outcomes 1 and 3 are interconvertable and produce a partial diploid 19 Lecture 8: Bacterial Gene Regulation Lac Operon* Regulation Positive (CAP) Negative (repressor) How mutations in each part of the operon would affect it Ex: two operons, one normal and one constitutively on If the mutation in the const on operon was in the repressor protein gene, the presence of the normal operon would suppress the mutation in the other operon because wild type repressor could bind to the promoter of the const on operon and begin regulating transcription. If however, the mutation is in the promoter, wild type repressor cannot bind and the operon is still const on See attached diagram for additional information Quorum Sensing (don't get caught up in the specifics, just understand the basic concept) Cells actually communicate with each other instead of living as isolates. They can sense cell density and change their expression patterns to account for the changes (eg. becoming competent for gene transfer, preparing for depletion of media nutrients in a high cell density) Attenuation Ex: gene for the enzyme that synthesizes tryptophan If the environmental levels of trp are low, there is no need for the cell to waste its energy synthesizing the enzymes to make trp since there is already trp present. However, if there is low environmental trp, the cell will need to make the enzymes to make trp. So the cell wants low expression levels in high trp conc and high expression levels in low trp conc. Two ways that E. coli accomplishes this: 1. A repressor protein that binds to the DNA in the presence of trp, physically inhibiting transcription. (Obviously it doesn't bind in the absence of trp.) 2. Attenuation o The cell doesn't directly monitor environmental levels of trp;instead it monitors conc of tRNA(trp) since high levels of environmental trp get incorporated into high levels of tRNA(trp). The mRNA of the genes encoding for the trp synthesizing enzymes are able to form two mutually exclusive secondary structures. o The placement of the ribosome on the mRNA directs which secondary structure forms. (Thus attenuation is dependent 20 on simultaneous transcription and translation, which is why eukaryotes don't utilize attenuation.) o Right before the gene for the enzyme that synthesizes trp is a gene for a short polypeptide (say gene X) o While the ribosome is translating gene X, it reaches a point in the mRNA where two trps are required (2 trps in a row is actually very rare in most proteins). o If the levels of tRNA(trp) are low, it takes a while for the 2 trps to be incorporated by the ribosome because there just isn't much trp to be had.  During this ribosomal delay, a mRNA secondary structure (antiterminator) forms that hides the stop codon for transcription and allows RNA polymerase to transcribe the trp synthesizing genes. o If the levels of tRNA(trp) are high, the ribosome has no trouble incorporating the 2 trps into polypeptide X and there is no pause. The ribosome quickly finishes translation of X and falls off of the mRNA.  This allows a different mRNA secondary structure (terminator) to form which exposes the stop codon to the RNA polymerase and stops transcription before the gene encoding the trp synthesizing enzyme can be transcribed. 21 Lecture 9: Drug Resistance and Transposons Mechanisms of Drug Resistance Bacteriocidal Kill bacteria, ampicillin Bacteriostatic Slow or stop bacterial growth-which resumes once drug is removed Drug Resistance Gene Classes 1. Enzyme alters drug structure-not the same compound- Ex: PCN-b-lactamase 2. Alteration of drug target structure Ex: Erythromycin-will put methyl group on binding site of 3. alteration of membrane behavior-drug pumps-molecs inserted into membrane, lowers intercellular concentration of drug 4. gene encoding drug-resistant form of enzyme-whole new gene takes over for one that would normally be present-creates a new pathway See other notes 22 Lecture 10: Mechanisms of Microbial Pathogenesis The Koch Postulates 1. The microorganism must be found in all cases of the dz 2. It must be possible to isolate the microorganism from the host and grow it up in pure cx 3. The microorganism must reproduce in the original dz when introduced into an experimental animal 4. The microorganism must be recoverable from the experimentally infected host Molecular Koch Postulates 1. Gene is present in strains of bacteria that cause the dz 2. Gene is absent in avirulent strains 3. Disrupting the gene reduces virulence 4. re-introduction of the cloned gene into the disrupted avirulent strain restores virulence Action of Select Microbial Toxins The classic virulence factor, which in purified form can damage the host A-B Toxins A. carries the toxin activity B. binds to the host cell Both encoded in a single polypeptide Clostridial Toxins 8 toxins from the two species listed below, all affect the same process Proteolytic toxins Botulinum A family of 7 related toxins ingested orally and enters the motor neurons at the neuromuscular jxn by binding to a receptor Causes flaccid paralysis by blocking acetylcholine release No muscular contraction Tetani Causes spastic paralysis by blocking inhibitory neurons in the CNS-failure to relax excited muscle After uptake, the toxin is taken into the body of the neuron, where it blocks release of inhibitory transmitters Cholera Toxin Also an A-B toxin One of the most common toxic mechanisms Involves ADP-ribosylation of host components involved in diverse processes Basic Rxn: adenosine diphosphate is transferred from Nicotinamide-adenosine diphosphate to a target protein CT (Cholera Toxin) Mobility Encoded in the chromosome vibrio cholera-has an element that looks like a transposon con
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