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129 Cards in this Set

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What are the two modes of regulation in a cell?
1. regulation of a pre-existing enzyme (posttranslational regulation)- inhibiting, modification, or degradation
2. regulation of amount of enzyme synthesized- transcriptional, mRNA stability, or translation
How does feedback inhibition work?
The end product of a biosynthetic pathway affects the activity of the first enzyme in the pathway.
What is an allosteric enzyme?
An enzyme that has two binding sites, one for the substrate, the other for the allosteric regulator.
What happens to an allosteric enzyme when the allosteric regulator binds to it?
The enzyme undergoes a conformational change, rendering it inactive.
What is a specific example of a feedback inhibition mechanism?
The synthesis of proline and arginine. It is a branched pathway, with both pathways using glutamic acid.
What are isozymes?
Isozymes are multiple enzymes that carryout the same reaciton. Each isozyme is independently regulated.
What is a specific example of a feedback regulation of isozymes?
The synthesis of DAHP is controlled by 3 isozymes, each one inhibited by one of 3 aromatic amino acids.
When would DAHP synthesis come to a complete stop?
An excess of all 3 amino acids is required to completely shut off DAHP synthesis.
How can an enzyme be regulated by covalent modification?
Covalently bonding certain chemical groups that can alter the enzyme's activity, usually rendering it inactive.
How are these modifications reversed?
Either by the same modifying enzyme (removing the chemical group) or a separate enzyme.
What is a specific example of enzyme regulation by covalent modification and how does it work?
The synthesis of glutamine synthetase-it is modified by adenylylation, which is the addition of AMP, which makes it inactive.
Which step during transcription is usually regulated?
The initiation step.
What proteins and enzymes does regulation of transcription usually involve?
It usually involves the sequence-specific interaction of a regulatory protein with DNA, RNA polymerase, or both.
What are the 2 types of modes of transcription regulation?
Positive and negative regulation.
What are activators?
They are regulators that stimulate transcription.
What are repressors?
They are regulators that inhibit transcription.
What are inverted repeats(IR)?
They are regions of DNA containing repeated sequences that are repeated in inverse orientation(TGTGTG---CACACA). They are usually the site at which proteins combine specifically with DNA.
What kind of proteins interact with IRs?
DNA binding proteins are usually dimers, most being homodimeric (composed of two identical polypeptides).
What part of the DNA binding protein binds with the DNA and where?
Each polypeptide in the dimer has its own domain, which interacts within the major groove of the DNA.
What are the 3 major structural classes of DNA binding proteins?
1. helix-turn-helix
2. Znic-finger
3. leucine zipper (coiled-coil)
Describe the helix-turn-helix structure.
It consists of a polypeptide that attains an alpha-helix secondary structure; this is the recognition helix that interacts specifically with DNA. The recognition helix is linked to 3 amino acids (the first one usu. being glycine and causing the "turn" in the protein). The other end of the "turn" is connected to the stabilizing helix, which stabilizes the recognition heliz by interacting hydrophobically with it. HTH proteins are often dimers and are common regulators in bacteria.
Describe the Zinc-finger structure.
The protein binds a Zn ion, which includes 2 cysteine and 2 histidine residues. Part of the "finger" of amino acids that is created forms an alpha-helix, and this recognition helix interacts with DNA in the major groove. There are typically at least 2 such fingers on the protein involved in binding. This type of protein is common in eukaryotic cells.
Describe the leucine zipper structure.
This structure contains regions in which leucine residues are spaced every 7 amino acids (like a zipper). The leucine zipper does not directly interact with the DNA, but it functions to hold 2 recognition helices in the correct orientation to bind DNA.
What is a specific example of a helix-turn-helix protein?
Lamba Cro
Through what kinds of bonds does DNA binding occur?
1. Hydrogen bonds
2. Ionic bonds
3. van der Waals interactions
What is negative control?
A regulatory mechanism that stops transcription.
What are the 2 types of negative control of transcription?
1. repression
2. induction
How does repression work?
Transcription for a specific enzyme only takes place when there is an absence of its substrate. An excess of the subsrate represses the synthesis of the enzyme. Enzyme repression usually involves biosynthetic/anabolic enzymes.
How does induction work?
An enzyme is made only when its substrate is present. Enzyme induction usually involves catabolic enzymes.
What is a specific example of enzyme repression?
Arginine biosynthesis. ArgR (repressor) is an allosteric regulator that only binds DNA when the corepressor (arginine) is bound. When arginine is present, the repressor binds to the DNA blocking transcription.
What is a specific example of enzyme induction?
The lactose operon. LacI is an allosteric protein that binds DNA in the absence of an inducer. When lactose is present, the inducer binds ot the repressor and transcription occurs.
What is positve control of transcription?
A regulator protein activates the binding of RNA polymerase.
What is a specific example of positive control of transcription?
The catabolism of maltose. MalT is an allosteric protein that binds DNA in the prescence of an inducer. When activator proetin and inducer bind to DNA, so does RNA polymerase.
How do activator proteins help RNAP bind to the DNA?
1. by altering the structure of DNA
2. direct contact between the activator and the RNAP complex (loop structure if activator binding site is hundreds of base pairs away from the promoter)
3. both combined
What is a regulon?
Numerous operons that are under the control of a single regulatory protein.
Give a specific example of a regulon.
Maltose utilization is encoded by a regulon. MalT plus its inducer bind to one of many activator-binding sites, which results in transcription.
What is global control?
Global control is when regulatory mechanisms respond to environmental signals and effect many different genes.
What is an example of global control?
Carbon utilization in bacterial cells. Some cells use selective mechanisms for the use of more efficient carbon sources when more than one is available.
What is catabolite repression?
When a preferred carbon source is available to E. coli, synthesis of enzymes needed for the use of the alternate carbon source are repressed by catabolite repression.
What is diauxic growth?
There are 2 exponential growth phases in the growth curve. E. coli use glucose first, and once that runs out, it uses lactose.
What kind of transcription control is the lac operon regulated by?
Both positive and negative control.
How is the lac operon positively controlled?
The CAP-cAMP is required for RNAP binding. CAP (catabolite activator protein) requires cAMP (cyclic AMP) as a co-activator. The binding site for RNAP is on the promoter.
How is the lac operon negatively controlled?
The LacI repressor binds to the DNA on the operator. When lactose is present, it acts as an inducer and renders the repressor inactive.
On what does the positive control of the lac operon depend?
The glucose levels in the cell. When levels are low, adenylyl cyclase is activated, and cAMP is formed, which then binds to CAP, and together the 2 bind to the promoter site on the DNA.
Would lac operon repsond in the following conditions?:
1. glucose, no lactose
2. lactose + glucose
3. lactose, no glucose
4. cyaA mutant (adenylate cyclase) + lactose
1. no transcription
2. no transcription until glucose ran out
3. transcription
4. no transcription
What is an operon?
A single transcriptional unit (a cluster of genes arrnaged in a linear and consecutive fashion) expressed coordinately from a single promoter and regulated by a common operator. The mRNA is usually polycistronic (genes encoding related enzymes are clustered together).
What is an operator?
A specific region of the DNA which is adjacent to the coding region of the first gene of the operon; it is located near downstream from the operon, near the promoter.
What is a regulon?
A set of operons, whose locations vary on the chromosome, are regulated by a common repressor or activator. Products of operons are often related, members of the same pathway.
What are alternative sigma factors?
Different sigma factors that recognize only a certain subset of genes in the genome. They can alter the specificity of promoter recognition of the RNA polymerase holoenzyme.
What is a two-component regulator?
A regulatory system containing two proteins: a sensor kinase and a response regulator.
What is a sensor kinase protein?
one of the 2 memebers of a two-component system; a membrane integrated protein that phosphorylates itself in response to an external signal and then transfers the phosphoryl group to a response regulator protein.
What is a response regulator protein?
The second member of a two-component system; it is phosphorylated by a sensor kinase
What is attenuation?
A regulatory process where the control occurs during transcription elongation. The number of completed transcripts from an operon is reduced, but the number of initiated transcripts is not changed.
What is a specific example of attenuation?
Tryptophan biosynthesis in
E. coli. The trp operon is controlled by the repressor protein TrpR and an attenuation mechanism.
What is the leader sequence?
It encodes for a polypeptide that contains tandem tryptophan codons near its terminus and functions as an attenuator.
What are 2 important features of the leader region?
1. RNA produced by transcription of the leader region has multiple and competing secondary structures (one of these structures is an intrinsic terminator sequence).
2. it encodes a small peptide that has codons for Trp.
This is key because the leader protein has codons related to the biosynthetic pathway encoded by the operon.
How do varying levels of tryptophan affect attenuation?
1. when there is excess Trp, the leader region will be synthesized and transcription of the remainder of the trp operon will be terminated.
2. when there are low levels of Trp, the leader region isn't synthesized and tracnscription of the rest of the trp operon takes place.
What are the 2 main categories of antimicrobial agents?
1. chemical agents (generally used topically or to clean inanimate object)
2. synthetics and natural agents (used in vivo)
What are the 3 ways antimicrobials work on microorganisms?
1. stop their growth (-static)
2. kill them without cell lysis (-cidal)
3. kill them by lysis (-lytic)
How would the viable cell count and total cell count for a bacteriostatic anitmicrobial agent be affected over time?
Both would cell counts would stop increasing, because there would be no more growth.
How would the viable cell count and total cell count for a bacteriocidal antimicrobial agent be affected over time?
The total cell count would stop increasing, but the viable cell count would decrease, because cells are dying.
How would the viable cell count and total cell count for a bacteriolytic antimicrobial agent be affected over time?
Both would decrease, because not only are the cells dying, but they are also lysing.
What are the 2 common methods for measuring how good an antibiotic is?
1. minimum inhibitory concentration (MIC)-a.k.a "tube dilution test"
2. agar diffusion method
What does the MIC test determine?
It determines the smallest amount of agent needed to inhibit the growth of a test organism.
How is the MIC test performed?
A series of liquid cultures is prepared and inoculated, with each tube containing a different concnetration of the agent. After incubation, the tubes are checked for visible growth (turbidity) and the first one (lowest concentration) that contains no growth at all is the MIC.
How is the agar diffusion method performed?
A Petri plate is prepared with a lawn of a given organism. Known amounts of the antimicrobial agent are added to filter paper disks, which are then placed on the surface of the agar. During incubation, the agent diffuses from the filter paper onto the agar.
What is a zone of inhibition?
The resulting absence of growth around the filter disks in an agar diffusion method. This test is commonly used to test for antibiotic susceptibility to pathogens.
What are the 3 classes of chemical agents used as antimicrobial agents?
1. antiseptics
2. disinfectants
3. sterilants
What are antiseptics?
Chemical agents that kill or inhibit growth of microorganisms and are nontoxic enough to be applied to living tissue (topically).
What are disinfectants?
Chemicals that kill microorganism (but not necesarily endospores)and are also used on inanimate objects.
What are sterilants?
Sterilants, also called sterilizers or sporicides, detroy all forms of microbial life, even endopores.
What is another name for natural antimicrobial agents?
What is a chemotheraputic agent?
Any chemical drug used to treat a disease caused by an invading organism (used interanlly).
What are chemotheraputic drugs classified by?
Their structure and/or mode of action.
What are some of the structures by which chemotheraputics drugs are classified (5)? Give an example of each.
1. carbohydrate-containing (Streptomycin)
2. macrocyclic lactones (Rifampin)
3. quinone-like (Mitomycin C)
4. amino acid and peptide analogs (Ceftriaxone)
5. heterocyclic compounds containing nitrogen (Polyoxin B)
What is the main target of chemotheraputic drugs that inhibit protein synthesis?
What is the main target of chemotheraputic drugs that inhibit cell wall synthesis?
Peptidoglycan synthesis.
What is a key feature of all chemotheraputic drugs and describe what it is?
Selective toxicity. It's the ability of an antimicrobial agent to be selectively more toxic to a microorganism than to animal cells.
What are 2 classes of synthetic antimicrobial drugs?
1. growth factor analogs
2. quinolones
How do growth factor analogs work?
They are structurally similar to a growth factor, but the subtle structural difference prevents the growth factor analog from functioning in the cell.
What is a specific example of a growth factor analog and how does it work?
Sulfa drugs, the simplest being sulfanilamide. It mimics p-aminobenzoic acid and blocks the synthesis of the vitamin folic acid (a nucleic acid precursor) by binding where p-aminobenzoic acid normally would.
How do quinolones work?
They interact with DNA gyrase and prevent it from supercoiling bacterial DNA, a step required for packaging the DNA in the bacterial cell.
What is a specific example of a quinolone?
Nalidixic acid. A fluoronated derivative of nalidixic acid, Ciproflaxin (Cipro), is used to treat urinary tract infections in humans.
What is an antibiotic?
Naturally-occurring antimicrobial agents. The term refers to compounds that are isolated from one living organism that kill or inhibit growth of other organisms. The term is loosely used as a synonym for more specific catergories such as antibacterial drugs.
What are the different types of activity that antibiotics can have (5)?
1. antibactierial
2. antifungal
3. antiviral
4. antiparasitic
5. anticancer
Why do most anibiotics that are discovered not have a medicinal use?
They are too toxic (lox selective toxicity).
Which kind of bacteria are more sensitive to antibiotics?
Gram positive.
What are broad-spectrum antibioitcs?
They affect both Gram positive and Gram negative bacteria.
What are narrow-spectrum antibiotics?
They only affect a single group of bacteria.
How do medicinally important antibiotics work?
They target cellular structures or machinery of bacteria but not of humans (e.g. cell wall, cytoplasmic membrane, or biosynthetic processes like transcription or translation).
What kind of antibiotics do we get from fungi?
Beta-lactam antibiotics.
How common are beta-lactam antibiotics?
They account for over 1/2 of all antibiotics produced and used worldwide.
What are the 2 "flavors" of beta-lactam antibiotics?
Penicillins (from Penicillum) and Cephalosporins (from Cephalosporium).
What was the first antibioic discovered? On which kind of bacteria did it work?
Penicillin G by alexander Fleming in 1929. It only worked on gram positive bacteria, and therefore had a narrow spectrum.
On which kind of bacteria do new generation penicillin derivatives work?
They have a broader spectrum, and are therefore called semi-synthetic antibiotics.
How do Cephalosporins differ from Penicillins?
They have small structural differences that allow the Cephalosporins to work on a broader spectrum of bacteria.
How do beta-lactam anitbiotics work?
They all inhibit cell wall synthesis.
What are 3 "flavors" of antibiotics that attack transcriptional apparatus in bacteria?
1. rifamycins
2. streptovaricins
3. actinomycin
How do rifamycins and streptovaricins work?
They attack the beta subunit of RNA polymerase. These antibiotics are specific for bacteria but also affect chloroplast and mitochondria at much higher concentrations.
How does actinomycin work?
It fits into the major groove of DNA and presents a road block for RNA polymerase.
What are the main sources of antibiotics?
Fungi or bacteria.
How do antibiotics that attack the translational machinery in bacteria work?
They interact with the rRNA and interfere with ribosome function and either inhibit translation initiation or translation elongation.
Which kinds of antibiotics are most medicinally useful?
Ones that target translational machinery. They are also widely used in research.
What is an example of an antibiotic that inhibits translation initiation?
What are some examples of antibiotics that inhibit translation elongation (3)?
Puromycin, chloramphenicol, and erythromycin.
What are 3 classes of antibiotics that we get from bacteria?
1. aminoglycosides
2. macrolides
3. tetracyclines
What are aminoglysides and how do they function? Give some examples.
They are amino sugars linked by glycosidic bonds. They inhibit protein synthesis by interacting with the 30S ribosomal subunit. they are effective against Gram negative bacteria, but they are not used much anymore and have been replaced by less toxic, more effective semi-synthetic antibiotics. Examples include streptomycin, kanamycin, gentamycin, and neomycin.
What are macrolides and how do they function? Give some examples.
They are lactone rings connected to sugars (sugar moiety). They inhibit the 50S ribosomal subunit in protein synthesis. Examples include erythromycin, spiramycin, and tyloson. Erythromycin is commonly used by people who are allergic to penicillin.
What are tetracyclines and how do they function?
they have a 4-ring structure. They inhibit protein synthesis by blocking the 30S subunit A site. They were one of the first "broad-spectrum" antibiotics discovered.
Which class of antibiotics have the most strains of bacteria that are resistant to it?
Tetracyclines (150 tons used). Streptomycin and kanamycin are 2nd and 3rd respectively (both more than 125 tons used).
What percent of N. gonorrhoeae strains were resistant to drugs in 1990?
What type of bacteria are most drug resistant strains?
Gram negative.
What are 3 categories of drug resistance?
1. natural or inherent resistance
2. mutational and secondary resistance
3. transferable resistance
Which category is of most clinical relevance?
Transferable resistance.
Define natural resistance.
Natural resisitance is chromosomally mediated and is predictable.
What are 3 reasons for natural resistance?
1. organism lacks the structure that the antibiotics inhibits (mycoplasmas lack cell walls)
2. organism is naturally impermeable to the antibiotic (Gram negative bacteria can't take up penicillin G)
3. organism is able to alter the antibiotic rendering it inactive
How are mutational and secondary resistance similar and how do they differ?
Both result from mutations, but secondary resistance occurs after therapy with the antimicrobial in question has begun.
What is the clinically importnat difference between mutational and secondary resistance?
The use of the drug may cause secondary resistance whereas mutational resistance is pre-existing. Also, antimicrobial therapy may select for resistant organisms. Normal organisms will be inhibited or killed by the therapy allowing resistant ones to prosper due to the lack of competition for food sources and advantageous environmetal niches.
What is transferable resistance?
It is the transfer of drug resistance from one organism to another by transfer of genetic information, which occurs by conjugation (plasmid-mediated transfer), phage-mediated transfer (transduction), or transformation.
What are the 3 mechanisms of resistance?
1. inactivation of the drug
2. efflux pumps
3. modify the target
What are some examples of inactivation of the drug?
1. enzymatic hydrolysis of the beta-lactam ring (cleaves bond) of penicillins and cephalosporins
2. acetylation of chloraphenicol
3. addition of various substituents to certain sites on the aminoglycoside antibiotics
What is the enzyme involved in the inactivation of chloramphenicol?
CAT-chloramphenicol acetyl transferase.
What are 3 enzymatic routes to aminoglycoside deactivation?
1. acetylation by acetyl CoA
2. phosphorylation by ATP (adding phosphate)
3. adenylation by ATP (adding AMP)
What are efflux pumps?
They are pumps used by bacteria to transport drugs out of their cytoplasm leading to insufficient concentration inside the cell to be effective.
From where do the efflux pumps obtain their energy?
Four protein subfamilies (all antiporters) get their energy from the EMF, and the ATPase family (ABC transporter) uses ATP.
How does the ABC transporter work?
The substrate binds to a dimer formed of two leaflets, which then causes a conformational change in the structure and the substrate is pushed out the top of the dimer, out of the cell.
What is modified in the modifcation resistance mechanism?
The target modifies itself.
Give a specific example of target modification.
Methylation of 23S rRNA by the methyltransferase ErmE causes ribosomes to become erthyromycin resistant.
From where do resistant mechanisms originate?
They originate from the organisms that produce the toxins. These organisms produce the toxins to eliminate competition in their habitat, but most protect themselves from the toxins. These mechanisms are then passed on genetically to other organisms.