BC EXAM 2 FULL STUDY GUIDE
Fungi
-Mycology: the study of fungi -Chemoheterotrophs (Decompose organic matter) -Aerobic or facultative anaerobic -Yeast vs. Mold -Grow better at pH of 5 -Grow in high sugar and salt concentration -Can grow in low moisture content -Can metabolize complex carbohydrates
List fungal diseases listed in lecture notes
-Mycosis: fungal infection -Systemic mycoses: deep within the body -Subcutaneous mycoses: beneath the skin -Cutaneous mycoses: affect hair, skin, and nails -Superficial mycoses: localized (e.g., hair shafts) -Opportunistic mycoses: fungi harmless in normal habitat but pathogenic in a compromised host -Ex: HIV> AIDS -immune compromised are at risk Opportunistic mycoses
List features of Chlamydia,
-No peptidoglycan in the cell wall; grow intracellularly Chlamydia and Chlamydophila -Form an elementary body that is infective -Chlamydia trachomatis causes trachoma and urethritis -Chlamydophila psittaci causes respiratory psittacosis -obligate intracellular bacteria -Elementary Body> Reticular Body -Elementary Body: small, small inactive, use for transmission> for disease -Reticular Body: Large, active, and inside human cells> causes some disease(eye infection, sex transmitted disease)
Cholesterol content
-OH group forms H-bonds with carbonyl of phospholipid head group and is buries the rest of it in the hydrophobic region of the phospholipid bilyaer. -Can form complex with specific phospholipids (sphingolipids); may increase rigidity (decrease fluidity) and order in regions of membrane (i.e. lipid rafts). -Fused ring structure is rigid; can disrupt regular interactions between phospholipid tails ---- increasing the membrane fluidity
Virus general structure
-Obligatory intracellular parasites -acellular -Contain DNA or RNA -Contain a protein coat -No ribosomes -No ATP-generating mechanism -no metabolism -can self replicate but need a host Rely on host for ribosomes,ATP-generating mechanism, metabolism, replication, transcription, translation, self replicating
Viral Syndromes
-Oral and respiratory tract infections -Flu-like and systemic symptoms -Exanthems and skin rashes -Infections of the eye -Hemorrhagic fevers -Gastroenteritis -Hepatitis -Infections of the central nervous system -Sexually transmitted viral diseases -Congenital, neonatal and perinatal viruses
Allosteric, what do we mean
"other shape" in greek sigmoidal kinetics (+/- cooperativity) conformational change-usually involves binding small effector molecules
Km
(K(-1) + K(2))/K(1)
synthetic DNA
-Artificial gene synthesis -DNA printing is a method in synthetic biology that is used to create artificial genes in the laboratory -Builds genes using a DNA synthesis machine
Multiplication of Animal Viruses
-Attachment: viruses attach to the cell membrane -Entry by receptor-mediated endocytosis or fusion -Uncoating by viral or host enzymes -Biosynthesis: production of nucleic acid and proteins -Maturation: nucleic acid and capsid proteins assemble -Release by budding (enveloped viruses) or rupture
What is plaque assay and what is used for?
-Bacteriophages form plaques, -which are clearings on a lawn of bacteria on the surface of agar
How Do Enzymes Decrease Activation Energy
-Bind the transition state of a reaction better than substrate -
List Scientific applications of recombinant DNA.
-Bioinformatics: understanding gene function via computer-assisted analysis -Proteomics: determining proteins expressed in a cell -Reverse genetics: discovering gene function from a genetic sequence -Southern Blotting:DNA probes detect specific DNA in fragments (RFLPs) separated by gel electrophoresis -Ti Plasmid:occurs in Agrobacterium tumefaciens Integrates into the plant genome and causes a tumorlike growth Can be used to introduce rDNA into a plant -DNA fingerprinting:D used to identify pathogens PCR microarrays and DNA chips can screen samples for multiple pathogens
vectors
-Carry new DNA to desired cells -Must be able to self-replicate -Plasmids and viruses can be used as vectors -Shuttle vectors exist in several different species and can move cloned sequences among various organisms
List theruputic applications of recombinant DNA
-Cervical Cancer Vaccine: consist of viral proteins; produced by Saccharomyces cerevisiae or by insect cells -Colony Stimulating Factor(CSF): counteracts effects of chemotherapy, improves resistance to infectious disease(Ex: AIDS), used in treatment of leukemia, produced by Eschericha coli and S. cerevisiae -Interferon alpha-leukimia, melanoma, hepatitis ( e. coli+ S.cerevisiae) beta-sclerosis(mammalian cell culture) gamma-chronic granulmatous disease(E. Coli) -Hepatitis B Vaccine: produced by (S .cerevisiae) carries hepatitis virus to plasmid -Human Growth Hormone(hGH): correct groeth deficiencies in kids(e. Coli) -Human Insulin: Diabetes, better than animal insulin( e. coli) -Influenza Vaccine: vaccine made from ( e. coli+ S.cerevisiae), carry virus gene -Tumor Necrois Factor(TNF): cause disinegration of tumor cell(e. coli) -Nanospheres: nao-particle, used fro drug targeting ** know that e. coli and S.cerevisiae(Yeast) are good host cells
List features of Spirochetes
-Coiled and move via axial filaments -spiral shape -filaments allow the bacteria to move -Treponema T. pallidum causes syphilis -Borrelia Causes relapsing fever and Lyme disease
What is meant by gene library?
-Collections of clones containing different DNA fragments( cut up by restriction enzm) -An organism's DNA is digested and spliced into plasmid or phage vectors and introduced into bacteria -At least one clone exists for every gene in the organism
Restriction enzymes
-Cut specific sequences of DNA -Destroy bacteriophage DNA in bacterial cells -Methylated cytosines in bacteria protect their own DNA from digestion -Create blunt ends or staggered cuts known as sticky ends
transcription
-DNA --> RNA -RNA polymerase binds to DNA at promoter region, then copies the template strand from 5' to 3' to mRNA (assembling nucleotides specific to RNA), then terminates when done and mRNA is released
differences between DNA polymerase and RNA polymerase
-DNA is in replication and synthesizes DNA, RNA is in transcription and synthesizes mRNA -DNA unzips DNA into 2 strands, RNA unzips DNA a few codons at a time and zips them back together -DNA requires the use of a primer to start (RNA primer made by primase), RNA does not -DNA proofreads systems and detects/removes errors, RNA does not
DNA
-DNA is the blue print for cell' protein, including enzyme -double helix -two strands anti parallel -deoxyribose sugar (5 carbon)(backbone)+ phosphate+nucleidotide(AT/GC) -DNA is obtained from parent cell during cell division -DNA can be expresses within a cell or translated to another through recombination and replication
List the enzymes involved in DNA replication
-DNA ligase -DNA Polymerase -Helicase *RNA Polymerase -Topismerase -Gyrase
anti-parallel
-DNA sides of the helix run opposite of each other, one is 5-3 one is 3-5 -also mirroring base pairs
prokaryotic vs eukaryotic
-DNA: in pro, 1-2 circular, sometimes linear. in euk, linear -histones: pro have in archaea, euk have always (organelles do not have though) -1st AA in protein synth: in pro, methionine for archaea and formylmethionine for bacteria. in euk, methionine -ribosomes: in pro, 70S. in euk, 80S (their organelles have 70S) -growth: in pro, binary fission. in euk, mitosis (organelles through bin fission)
Diversity Within the Archaea
-Distinct taxonomic grouping; lack peptidoglycan -Extremophiles(survive extreme condition) Halophiles Require salt concentration > 25% ( high salt) Thermophiles Require growth temperature > 80 C (high temp) hyperthermophile above boiling point -Methanogens(CH4) Anaerobic and produce methane -methan and CO2 are green house gases and can caouse global warming -but they are balanced by bacteria
ES Complex Assumptions
-ES complex is in rapid equilibrium with free enzyme -BREAKDOWN of ES to form products is assumed to be slower than the formation of ES and the breakdown of ES back to E + S
where to find DNA in eukaryotes and prokaryotes
-EUK: found in chromosomes in the nucleus, multiple, linear, diploid or haploid, contains 10,000s of genes -PROK: found in the chromosomes (bacteria's is in the plasmids), single, circular pieces, 1000s of genes
The Three Domains
-Eukaryotes originated from infoldings of prokaryotic plasma membranes -Endosymbiotic bacteria developed into organelles( mitochondria, chloroplast) -one cell engulfed the other
Describe the role of Mammalian cells in making gene products
-Express eukaryotic genes easily -Can make products for medical use Harder to grow, hard to work with
Describe the role of Plant cells in making gene products
-Express eukaryotic genes easily -Plants are easily grown, large-scale, and low-cost
Animal defenses against viral infection
-Fever(increase temp>kills virus) -Interferon(protein produced by immune system(unique to vrius) -Antibodies(protein broduced by immune system for many bacterias) -Examples of Successful Antiviral Drugs:(not many) -Reverse Transcriptase( prevent virus from making RNA from DNA) -Inhibitors against HIV -Protease inhibitors against HIV(inhibit protein from virus) -Amantadine treats influenza virus (flu) -Tamiflu: attack influenza virus (flu) -Acyclovir prevents HSV infections -Ribovirin: for HBV and HCV
Why you always need host to grow virus
-For a virus to multiply: -It must invade a host cell -It must take over the host's metabolic machinery
The Anoxygenic Photosynthetic Bacteria
-Green sulfur: phylum Chlorobi -Green nonsulfur: phylum Chloroflexi -Purple sulfur and purple nonsulfur photosynthetic bacteria are proteobacteria -Carry out anoxygenic photosynthesis
How different types of radiation may cause mutation
-Ionizing radiation (X rays and gamma rays) causes the formation of ions that can oxidize nucleotides and break the deoxyribose-phosphate backbone (DNA) -UV radiation causes thymine dimers (fuse two thymine together) Repair the Damage by (can' repair DNA): Photolyases separate thymine dimers Nucleotide excision repair: Enzymes cut out incorrect bases and fill in correct bases
Latent Viral Infections
-Latent virus remains in asymptomatic host cell for long periods and produces diseases -May reactivate due to changes in immunity Cold sores, shingles -No symptoms during latency, viruses not usually released
components of a virus
-MUST have capsid (protein shell) and nucleic acid (nucleocapsid) -can have envelope/spikes
Phosphorylation other details
-Many proteins are phosphorylated on multiple sites -Addition of a phosphate to a protein is a covalent bond -This is reversible through the action of phosphotases -Activity may be increased or decreased (specific to enzyme)
products of replication, transcription, translation, reverse transcription
-replication: 2 new DNA that are semi-conservative and IDENTICAL -transcription: RNA (& the mRNA is complementary to one strand of DNA) -translation: proteins -reverse translation: DNA
RFLP
-restriction fragment length polymorphism -finds differences in homologous DNA -DNA sample is broken into pieces by restrictive enzymes and the fragments are separated according to their length -genome mapping, localizing genes for genetic disorders, determining risk for disease
RNA
-ribonucleic acid -singe strand -5 carbon ribose sugar+ phosphate+nucleidotide(AU/GC) -RNA is read by ribosome to make protein
economic effects of fungi
-saccharomyces cerevisiae: bread, wine, hepatitis B vaccine -trichoderma: cellulase -taxomyces: taxol -entomophaga: biocontrol -coniothyrium minitans: kills fungi on crops -paecilomyces: kills termites
Extra-Chromosomal DNA (Plasmids, etc)
-self-replicating circular pieces of DNA -Carry genes such as antibiotic resistant genes or -May code for proteins that enhance the pathogenicity of a bacterium (disease causing genes)
Viroid
-short pieces of naked RNA Cause potato spindle tuber disease
genome
-sum of total genetic material for an organism -cells only have DNA genome -viruses have DNA or RNA genomes -they vary in size
rRNA
-synthesizes machinery of the cell in TRANSLATION -makes up ribosome (and proteins), which reads the codon from mRNA and puts the aminos together (after getting them from tRNA) w/ their base pair -the ribosome sits on mRNA, zips through it, reads codons, pairs them with aminos
plaque assay
-technique where clear patches where virus-infected cells have been destroyed; helps determine the quantity of infection/viral harm -dilutions of the virus are used to infect a cultured monolayer, covered with agar -the number of plaques directly relates with the number of infectious virus particles added to the plate
catabolite repression
-the glucose effect -stopping synth of enzymes involved in catabolism of C sources other than the preferred one -first initiated by glucose -when glucose is available, level of cAMP in cell is low, and thus CAP (protein involved in the metabolism of sugars) is not bound
metagenomics
-the study of genetic material directly from environmental samples -Human Genome Project sequenced entire human genome and will map proteins expressed in human cells
obligate intracellular pathogens
-this means they cannot multiply unless they invade a host and instruct its genetic/metabolic machinery -always need a host to grow and have host specificity -infectious agents that are included: Rickettsia and chlamydia -viruses can affect ALL organisms: humans --> bacteria and archaea
double helix
-twisted ladder of DNA with alternating "rungs" downward b/w sugar and phosphate -the sugar is deoxyribose, a 5 C sugar -the nucleotides (sugar, phosphate, base pair) are connected by H bonds
protozoa
-unicellular eukaryotes -inhabit soil and water -animal-like nutrition -complex life cycle -resistant to elements -cyst vs. trophozoite: cyst is hardy and is ingested via contaminated food/water, produces 2 trophozoite which multiply -some are human and animal pathogens: amebae, malaria, giardia, cryptosporidium
Ames Test
-use of bacterial genes to help screen for carcinogens -looks for damage to bacteria's DNA by looking for metabolic activity being affected by the mutation -reversions: new mutations that reverse the effects of the original mutation (ex: turning - bacteria to + bacteria at a higher rate than spontaneously)
recombinant DNA and insulin
-used to treat diabetes -better tolerated than insulin extracted from animals -produced be E. Coli
acellular structures
-virion: fully formed virus that can establish infection and affect anything -viroid: infectious pathogen that is a single stranded RNA with no protein coat; affects plants -prion: made of 2 proteins PrP; 2 forms: ALPHA helix (natural form) and BETA (disease causing form). accumulation of PrP causes neuro degeneration, mad cow disease, CJD in human, Kuru in sheep. hard protein=hard to change, must burn.
lysogeny
-where the host chromosome carries bacteriophage DNA; special (temperate) phages are inserted into bacterial chromosome called prophage -host cell replicates its chromosome and also replicates prophage DNA -results in phage conversion: host cell exhibits new properties -dangerous in human disease because phage genes cause toxins and enzymes to be produced -cancer causing viruses and animal viruses can undergo this
forms of fungi:
-yeast: nonfilamentous and unicellular -budding yeast: divide unevenly -fission yeasts: divide evenly -dimorphic fungi: yeastlike at 37 C and moldlike at 25 C -mold: multicellular
Stages of the catalysis of chymotrypsin
1) Acylation of the enzyme 2) Hydrolysis of the acyl-enzyme (deacylation)
Premises of the concerted model
1) Allosteric enzymes have several active sites on different polypeptide chains. 2) The enzyme can exist in two distinct conformational states, R and T. 3) No hybrid R/T states of the enzymes can exist, e.g. there are no enzymes with one subunit in the T and three in the R. 4) The substrate binds more readily to the R form of the enzyme than to the T.
Functional Properties of Enzymes
1) Biological Catalyst 2) Decrease Activation Energy for a RXN 3) DO NOT alter ΔG°' or Keq
Magnitude Change in G Depends on...
1) Chemical reaction itself 2) How far the system is from equilibrium 3) [reactants], [products], and number of bonds broken and made
Why is Hydrolysis Favorable
1) Reduced charge repulsion, going from 4 to 3 negative charges so the cell wants this to happen 2) Change in entropy - resonance forms in organic phosphate and resonance is more stable
Types of cofactors
1) Small organic molecules derived from vitamins 2) Metals
To understand how enzymes operate
1) The free energy difference (Delta G) between the products and the reactants 2) The free energy required to initiate the conversion of reactants into products
Chemical Reactions Raise 2 Questions
1) What will happen? 2) How fast will it happen?
Thermodynamic Properties of a Reaction
1) Whether they happen at all (Delta G) 2) What is the free energy required to initiate the conversion of products to reactants
How Do Enzymes Decrease Activation Energy
1) binds and orients substrate (positioning it is very important) 2) Strains/distorts bonds in the substrate 3) The binding energy (multiple weak interactions between enzyme and substrate as a driving force for catalysis) 4) Provides a micro-environment and increases collisions
Phosphorylation what 3 things happen
1) introduces negative charges 2) disrupts or forms electrostatic charges 3) can form H bonds
Pathways of Multiplication Used by Various RNA-Containing Viruses
1. Attachment 2. Entry and Uncoating(uncoating release viral RNA and roteins) 3. RNA replication by viral RNA dependent RNA polymerase 4.Transaltion and sythesis of viral proteins 5Maturation and release
Replication of a DNA-Containing Animal Virus
1. Attachment:Virions attach to host cells 2. Entry and Uncoating: Virions enter a cell and in DNA is uncoated 3.A portion of viral DNA is transcribed,producing mRNA that encodes early viral protein 4. Biosynthesis: Viral DNA is replicate and some viral proteins are made 5.Late translation; capsid proteins are synthesized 6Maturation: Virions mature 7: Release: Virions are releases *** important for drug development and understanding disease pathology *** can help stop virus from damaging anything
The Flow Genetic Material
1. Expression( cell metabolize and grows) -genetic info is used within a cell to produce the proteins needed for the cell to function ( Translation, Transcription) -add variety 2. Recombination (Recombinant Cell) - gentetic info can be transfered horizontally btw cells of the same generation -add variety 3.Replication (offspring of cells) -genetic info can be transferred vertically to the next generation of cells
Lysogenic Cylce( lambda) steps
1. Phage attaches to host cell and injects DNA 2.Phage DNA circulizes and enters lytic cylce or lysogenic cycle 3.Phage DNA integrates within the bacterial chromosone by recombination becoming a prophage 4. Lysogenic bacterium reproduces normally 5. Occasionally, the prophage may excise from the bacterial chromosome by another recombinanation event initiating a lytic cycle * if host make more virus= induction -No cell death, virus production, host death -virion reased from 1 to 100
Lytic Cycle Steps
1. Phage attaches to host cell and injects DNA (ATTACHMENT) 2.Phage DNA circulizes and enters lytic cylce or lysogenic cycle(PENETRATION) 3. New phage DNA and proteins are synthesized(ABSORBTION) 4. assembled into viroins(MATURATION) 5.Cell lyses, releaseing phage virions(EXIT)
Characteristics of Helminths
-Parasitic worms -Multicellular eukaryotic animals -Specialized to live in hosts May lack digestive system Reduced nervous system Reduced or lacking locomotion Complex reproductive system -EX:Namatodes effets human plants, pin worms
Polymerase Chain Reaction (PCR)
-Process of increasing small quantities (amplifying) of DNA for analysis -Used for diagnostic tests for genetic diseases and detecting pathogens -Reverse-transcription PCR uses mRNA as template
Prions
-Proteinaceous infectious paericles -Two stable forms of PrP Normal functional structure is α-helices Disease-causing form with β-sheets called prion Prion PrP converts cellular PrP into prion PrP by inducing conformational change Accumulation of prion PrP results in fatal neurological degeneration, deposition of fibrils in brain, and loss of brain matter -Inherited and transmissible by ingestion, transplant, and surgical instruments -Spongiform encephalopathies Mad Cow Disease CJD in human
Classification of Eukaryotes
-Protista: a catchall kingdom for a variety of organisms; autotrophic and heterotrophic Grouped into clades based on rRNA -Fungi: chemoheterotrophic; unicellular or multicellular; cell walls of chitin; develop from spores or hyphal fragments -Plantae: multicellular; cellulose cell walls; undergo photosynthesis -Animalia: multicellular; no cell walls; chemoheterotrophic
translation
-RNA --> protein synthesis -mRNA is the template -tRNA brings the AA to the P&A sides of the ribosome -ribosome sits on mRNA, zips through it, and matches the codons to pair with their aminos -as aminos build up, the bigger subunit of the 70S ribosome start shifting -peptide bonds form between tRNA -polypep chain reaches STOP codon and leaves to be folded into protein
tools in recombinant DNA
-RT (converts RNA to DNA) -synthetic nucleic acids (to make DNA in the lab) -restriction enzymes (to cut DNA into desirable lengths) (helps to study DNA separately in gel) -vectors (carry DNA into new host, i.e. plasmids or viruses)
ΔG° Equation
-RTlnKeq Depends on the constant because standard conditions and then the natural log of the Keq from the products and reactants SOOOO if we can determine the concentration of products and reactants at equilibrium, we can use this to determine Keq an then determine the change in free energy for conversion of one mole of reactant to product (s)
How does virus enter into host cells
-Receptor mediated endocytosis -fusion
Economic Effects of Fungi
-Saccharomyces cerevisiae(yeast): bread(make dough fluffy), wine(use corn for alchohol> fuel(made by yeast)), hepatitis B vaccine -Trichoderma: cellulase use enzyme to break down cellulose s other organism can use them -Taxomyces: taxol -Entomophaga: biocontrol some produce chemicals to help control insects -Coniothyrium minitans: kills fungi on crops -Paecilomyces: kills termites
What is the significance of Ti Plasmid
-Ti plasmid: occurs in Agrobacterium tumefaciens -Integrates into the plant genome and causes a tumorlike growth -Can be used to introduce rDNA into a plant 1. The plasmid is removed from bacterium, and the r DNA is cut by a restriction enzyme 2. Foreign DNA is cut by the same enzyme 3. The foreign DNA is inserted into T-DNA of plasmid 4. the plasma is reinserted into a bacterium 5.The bacterium is used to insert T-DNA carrying the foreign gene into the chromosome of the plant cell 6.The plant cells are growth in culture
What are the general features of Protozoa?
-Unicellular eukaryotes -Inhabit water and soil -Animal-like nutrition -Complex life cycles -Cysts vs. Trophozoite -Some are Human and animal pathogens Amebae(Blood Diarrhea, water borne disease) Malaria-Insect borne disease, in water), red blood cell, liver cell Giardia(Diarrhea, outdoor) Cryptosporidium(Diarrhea, Water, can cause outbreak) ** in water, use ozone to kill cyst, chlorine cannot kill cyst
What is the importance of Algae
-Unicellular or filamentous photoautotrophs -Lack roots, stems, and leaves -Mostly aquatic Water is necessary for growth and reproduction(make biomass from carbon dioxide) -Fix CO2 into organic molecules -Produce 80% of Earth's O2(oxenate planet) -Algal blooms are increases in planktonic algae that can result in toxin release or die and consume oxygen(overgrow) -Kill aquatic animals by suffocating them(taking oxygen) -Oil production -Symbionts of animals -Primary Producer for Oxygen
What is CPE? Give an example
-Virally infected cells are detected via their deterioration, known as the cytopathic effect (CPE) -Continuous cell lines are used
Describe three ways in which viruses are cultivated.
-Viruses must be grown in living cells -Bacteriophages are grown in bacteria -Bacteriophages form plaques, which are clearings on a lawn of bacteria on the surface of agar -Each plaque corresponds to a single virus; can be expressed as plaque-forming units (PFU) -In living animals -In embryonated eggs Virus injected into the egg Viral growth is signaled by changes cell culture -Tissues are treated with enzymes to separate cells -Virally infected cells are detected via their deterioration, known as the cytopathic effect (CPE) -Continuous cell lines are used
complementary strands
-a strand with the complementary base pairs to the template strand -can be the copy of DNA (cDNA) made from the RNA with the help of RT -C-G A-T U-A
operon
-a unit made up of linked genes that is thought to regulate other genes responsible for protein synthesis -inducible genes: controles catabolic processes- usually off unless something for them to metabolize; sugar metabolism; lactose operon -repressible genes: control genes involved in synthesis; anabolism and synthesis of AA; always on; tryptophan operon
PCR
-amplify DNA w/ out culture or purifying techniques 1. denaturation: heat and cool to separate DNA 2. priming/annealing: adding primers to ensure it will bind to DNA. primer indicates where rep/amp will begin 3. extension/elongation: DNA poly adds nucleotides to produce 2 new strands of DNA
exonuclease/exonuclease activity(+/-)
-an enzyme that breaks down a nuc acid by removing successive nucleotides ADDING -when inc base pair is recognized -DNApolyIII can't start synth, but keeps adding to existing chain in 5-3 direction- primase jump start replication -est 750 bases/second -palindromes: base sequences of endonucleases that are subject to identification/clipping REMOVING -DNA proofreading: something inc seen, DNA poly moves in 3-5 to fixed by removing inc base pair -these enzymes cleave DNA at desired sites
Transposons
-are segments of DNA that can move from one region of DNA to another -Contain insertion sequences (IS) that code for transposase that cuts and reseals DNA -Complex transposons carry other genes (e.g, in antibiotic resistance)
explain how base analogs may induce mutation
-cause mistake in base pairing and then it is copied wrong -randomly incorporated into cellular DNA in place of normal base -EX: antiviral or antitumor drugs -AZT azidothymine> HIV
chromosome
-cell structure composed of neatly packed DNA that carry hereditary info -chromosomes contain genes
eukarya
-cell wall varies in composition; contains carbs -membrane is composed of straight carbon chains attached to glycerol by ester linkage -1st AA in protein synth is methionine -contains common arm of tRNA
monolayer
-cells grow at the bottom of a flask; you can see when the virus kills them bc they disperse/detach and come off the flask -take liver cells and homogenize them and they become a monolayer -mix liver w/ cancer cells to make them an immortal cell line and continue transferring them to new flasks -areas where virus-infected cells have been destroyed show up as clear patches called plaques
base analogs
-chemical agents that look like a base so they fit into DNA, but doesn't function like one -sub for a normal nucleobase but is an incorrect nucleotide -form of point mutatin -ex: caffeine -can induce mutation by either affecting the process of replication or the structure of DNA
Carcinogens
-chemicals that can cause mutation and can cause cancer
carcinogen
-chemicals/radiation/etc that mutate DNA and cause cancer -mutate critical cell targets that regulation cell division -can occur in any tissue -Ames Test is used to screen for carcinogens
spirochaetes
-coiled and move via axial filaments -treponema: T. pallidum causes syphilis -borrelia: causes relapsing fever and lime disease
CPE
-cytopathic effects: visual damages caused by a virus -when the cell lysis and disintegrates, bc it its visible under the microscope -you can see the cells dying -inclusion bodies: compacted mass of viruses or damaged cell organelles in the nucleus and cytoplasm ex: common CPE- synctia; one large cell with multiple nuclei bc multiple host cells were fused together -also, the fusion of mult host cells into a single large cell containing mult nuclei
replication
-each half of DNA is a template, 1 parent DNA --> 2 new DNA -semiconservative replication: half is parent strand, half is new strand -DNA polymer III (origin of replication) elongates only from 5' --> 3' -leading strand: made into a completely new strand bc synthesis goes from 5' to 3' -lagging strand: 3' to 5' that is added in the opposite direction; okazoki fragments (short frags) are attached to the growing end of it -replication fork
genetic material for viruses, DNA or RNA, single stranded or double stranded helix
-either DNA or RNA, either SS or DS -more often SS RNA
reverse transcriptase
-enzyme that uses RNA to make DNA (or complementary DNA- cDNA) -unique to some viruses- like HIV -known for role in replication of AIDS -turns RNA back to DNA -tool in recombinant technology (bc recom, can only be used with DNA) -they are encoded by retroviruses, a special type of RNA virus
fungi
-eukaryotic -chemoheterotrophic (decompose organic matter) -single or multicellular -cell walls of chitin -develop from spores or hyphal fragments -aerobic or facultative -mycology: study of fungi -yeast grows in single cell, mold grows in multicell -grow better at pH of 5, high sugar & salt conc -can grow in low moisture content -can metabolize complex carbs
induced mutations
-exposure to mutagens - chemical or physical agents that interact with DNA in a disruptive manner -radiation/chemicals -ionizing radiation: forms ions and free radicals that do incorrect base substitutions or break the sugar phosphate backbone -UV radiation: causes bonding b/w adjacent thymine (forms thymine dimers). repair this by photolyases, which separates dimers, or nucleotide excision repair which cuts out incorrect bases and fills in correct
tRNA
-fishes in aminos from the outside of the cell to the P&A sides of the ribosome -contains anticodons
gene
-fundamental unit of heredity responsible for the given trait of an organism -a site on a chromosome that provides info for certain cell function -contains a necessary code for what to synthesize -specific segment of DNA
how to grow viruses in a lab
-get a host -pour into bacteria agar plate and see where bacteria was killed (plaques) -monolayer w/ immortalized cells lines and chkn embryo w/ whole organism
cell/tissue culture
-growing isolated cells in culture -bacteriophages also produce plaques when grown in agar cultures of their host cells; this is a way to count/detect bacteriophages -in vitro cultivation: animal cells cultured in a lab -tissue culture: tissues are treated with enzymes to separate cells; virally infected cells are detected due to their deterioration (CPE- cytopathic effect); continuous cell lines are used
Simultaneous Transcription and Translation
-have multiple ribosome making protein and transcribing RNA -Speeds up growth -Longer in Human than bacteria beccause RNA is in the nucleus than take to chloroplast and than have RNA sythensized
metagenomics,
-is the study of genetic material directly from environmental samples -The Human Genome Project sequenced the entire human genome -The Human Proteome Project will map proteins expressed in human cells
Characteristics of a transition state
-is unstable; difficult to isolate -distinguishes it from a true reaction intermediate. - Instability is due to the strain in the covalent bonds of the original reactant (in order to convert reactant to product). - Enzyme binds the transition more tightly than substrate because the transition state is unstable. • These additional noncovalent interactions result in maximal binding energy and release of that energy
shapes of viruses
-isocahedron: 3D, 20 sided figure with 12 evenly spaced corners - naked: geometric capsid assembled around a nuc acid strand -enveloped: virus composed a nucleocapsid surrounded by a flexible mem called an envelope -helical: have rod-shaped capsomers that form a series of hollow discs -complex: take shapes that are not symmetrical; never enveloped; UNIQUE to bacteriophages; head is icosahedral w/ helical shaped tail
latent virus
-latent: the virus remains unnoticed in host for a little, chronic -activates rapidly rarely then disappears -virus becomes a part of the host (maybe permanent) -ex: herpes, chkn pox
lysogenic virus
-lysogenic: bacteriophage infection- virus DNA becomes a part of the host cells DNA -bacteriophage: virus that infects bacteria -temperate phages: don't kill the host bacteria, but integrate their chromz into host's chromz -prophage: genetic material of the bacteriophage that is transmitted to daughter cells in replication -DOESN'T lyse the cell, makes it seem normal
what are ribosomes made of/types of rib in PRO and EUK
-made of rRNA and proteins -PRO: 70S- made of 50 big and 30 small subunits -EUK: 80S- made of 60 big and 40 small subunits -thus, antibiotics can target ribosomal ribosomes but not human
mRNA
-makes/carries a codon for the AA to make protein -provides direction for putting AA together -contains codons
mutation, nonsense mutation, frameshift mutation-
-mutation: permanent, inheritable changes in genetic information -nonsense mutation: a premature stop -frameshift mutation: when one or more bases are inserted or deleted in DNA strand. results in nonfunctional protein because every subsequent AA is different than original code. most serious.
mycoplasma
-mycoplasmateles lack a cell wall; pleomorphic (can alter shape and size based on environ conditions) -M. pneumoniae: causes mild pneumonia
fungal diseases
-mycosis: fungal infection -systemic mycosis: deep within the body -subcutaneous mycosis: beneath the skin -cutaneous mycosis: affects hair, skin and nails -superficial mycosis: localized (ex: hair shafts) -opportunistic mycoses: fungi harmless in normal habitat but pathogenic in a compromised hose
Describe how DNA can be inserted into a cells; By: Microinjection
-need a glass micro pipette with a diameter much smaller than the cell -the micro pipette punctures the plasma membrane and the DNA can be injected through it -insert DNA coated" Bullets" into a cell
chlamydia
-no peptidoglycan in cell wall, grow intracellularly -chlamydia and chlamydophila: form elementary body that is infective -chlamydia trachomatis: causes trachoma and urethritis -chlamydophila psittaci: causes respiratory psittacosis
viruses
-obligatory intracellular parasites (require living host cells to multiply) -contain DNA or RNA -contain a protein coat -no ribosomes -no-ATP generating mechanism
What causes spontaneous mutation/induced mutation
-occasional mistake in DNA replication -missing a mutation causing agents (not from a mutation)
Ti plasmid
-occurs naturally in the bacterium agrobacterium tumefaciens, which infects plants, allowing Ti plasmid to a crown gall (tumor-like growth) -T-DNA then integrates into genome of infected plant and stimulates crown gall while causing the production of certain products that the bacterium uses as a source of nutritional C and N -this provides a vehicle for introducing rDNA into a plant by inserting foreign genes into T-DNA, which will be put into a plant cell and hopefully create a new plant that expresses the foreign gene
persistent virus
-persistent: slow, long lasting infection -one form is chronic -viruses keep producing, but the cell doesn't die
Define lysogeny and describe its role in passing disease causing genes from one bacteria to another. What other genes may be exchanged using lysogenic viruses.
-phage remains latent -Phage DNA incorporates into host cell DNA Inserted phage DNA is known as a prophage When the host cell replicates its chromosome, it also replicates prophage DNA Results in phage conversion—the host cell exhibits new properties specialized transduction
Ames Test
-procedure using bacteria to identify potential carcinogens -The Ames test exposes mutant bacteria to mutagenic substances to measure the rate of reversal of the mutation -Indicates degree to which a substance is mutagenic
importance of cyanobacteria/algae
-produce 80% of the worlds O2 -fix CO2 in organic molecules -produces and secretes carbs -oil production -symbionts of animals
gel electrophoresis
-produces a readable pattern of DNA fragments (which were cut by restriction enzymes) -used to size/compare DNA frags -elec current on both sides of gel-DNA is neg so they run down to the pos charge -large slow small fast
bacteria
-prokaryotic -cell wall contains peptidoglycan -cell wall is made of straight C-chains attached to glycerol by ester linkage -first AA in protein synth is formylmethionine -sensitive to antibiotics -contains rRNA loop -contains common arm of tRNA
acrhaea
-prokaryotic -variable cell wall that contains NO peptidoglycan -membrane made of branched C-chains attached to glycerol by ether linkage -1st AA in protein synth is methionine
how to put DNA into host cell/how to track DNA
-put genes into bacteria/host via plasmids/virus -make cell competent (by treating it with hot/cold shock) so that it randomly picks up DNA from the environment -track through the monolayer or cell tissue cultures -genomic libraries: "a collection of isolated genes maintained in a cloning host"; collections of DNA clones that represent the genomes of many diff orgs; made by putting fragments of chromosomes into a set of bacteria
3.Name different RNA molecules. What is the role of each molecule? Codon, anticodon?
-rRNA -mRNA: codon -tRNA: anticodon
spontaneous mutation
-random change in DNA that arises because of errors in the base pairing of nucleotides in the old/new strands of DNA -caused my errors in replication that occurred randomly; NOT exposed to mutagens -cell repair system corrects many of these -ex: point mutation
recombinant DNA
-recombination gene tech: removing genes from one org and combining/replacing it with genes from another (only done with DNA, so RT is involved) -done to: 1. eliminate undesirable traits 2. create beneficial traits 3. create organisms that help humans' needs -clones are made to made many copies of genetically identical cells which all carry copies of the vector-which is an agent that carries and transmits an infectious pathogen into another organism (and thus the gene of interest)
ONE-STEP growth cycle
1. addition/penetration of viruses to cells, 1 hour period of absorption, no visible virus 2. eclipse phase for 10-12 hours virus becomes uncoated (no virus detected) 3. synthetic phase (phage particles), new virus particles are assembled, preformed components 4. latent period, production reaches max
order of virus life cycle
1. attachment- capsid/spikes 2. penetration- inject DNA/RNA 3. un-coating (possibly)- if capsid goes in 4. synthesize- replication and protein synthesis 5. assembly- put things together to make virus 6. release- cell must be ruptured, lysis of host (100x viruses made)
bacteria and viruses
1. bacteria= have 1,000s of genes, use own system and use host for protection only 2. viruses= have 100s of genes, acellular, electron microscopy
examples of virulence factors
1. enzyme: used to breakdown host barrier 2. structure: attachment via pili and fimbrae, capsule and glycoclax against phagocytosis 3. toxins: exo/endo 4. ability to grow and multiply
animal defenses against viral infection
1. fever- generates heat to fight infection and speed up immune system 2. interferon- antiviral proteins that affect RNA viruses 3. antibodies- proteins produced by immune system to recognize viruses and neutralize them
enzymes involved in DNA replication
1. helicase- opens and unzips DNA helix 2. primase- primer used to start replication 3. DNA polymerase- syntheszies DNA 4. Topoisomearse- used to bring back into coil shape 5. Ligase- fills gaps and fuses things together
PCR steps
1. incubate, seperate strand 2.add primers, nucleotide (denature 94) and DNA polymerase (Annealing 60) 3.incubate, all primers to attach to single strand DNA (extension 72) 4. incubate, DNA polymerase copied the target DNA at this temperature 5.Repeat cylcle of heating and cooling to make 2 more copies of target DNA -each cycle the number doubles
lysogeny
1. lysogenic cells cannot get infected by the same virus but are susceptible to other viruses 2. the virus genes get turned on and new traits introduced through specialized transduction 3. viral latency 4. cellular transformation
exceptions to Koch's postulates
1. polymicrobe diseases; diseases caused by more than one microbe 2. not all diseases are caused by microbes 3. not possible to isolate in pure culture some infectious agents
tools in recombinant DNA
1. reverse transcriptase= converts RNA to DNA 2. synthetic nucleic acids 3. restriction enzymes=cuts DNA at certain points 4. vectors= plasmids and viruses, introducing DNA to the host cell 5. gene library= collection of bacteria that each carry portions of DNA (collection of segments of DNA from host), tracking DNA
ports of entry for infectious agents
1. skin (ex. staph/strep) 2. mucous membrane (ex. respiratory tract-crytoccocus, digestive tract, urogenital tract-HIV, GI tract-e.coli) 3. parenteral route (ex. needle, cut, insect bites, bruises, surgery)*most common 4. placenta
Rate enhancement of carbonic anhydrase
1.0*10^6 molecules/second * 1/(1.3*10^-1 molecules/second) = 7.7*10^6
MECHANISMS OF VIRAL PATHOGENICITY; Cytopathic Effects (CPE)
1.Ability to enter the cell (penetration) Directly via trauma or insect bites Through mucous membrane (respiratory, GI, or Urogenital tracts) 2.Ability to multiply within the cell and to evade host immune system 3.Ability to damage the host; Cytopathic Effects (CPE) Examples of Cytopathic Effects (CPE) Cell lysis or transformation (plaque formation)Altering Nuclear and or Cytoplasmic structures Changes in cell membrane elasticity, result in fibroblast formation and viral budding Take control of host gene expression and metabolism Inhibits host protein synthesis/RNA synthesis Encourage host cell apoptosis (self lysis) Cell fusion, syncytia formation
triangle of infection
1.microbe- number and growth rate affect infection 2. host- LD50 and ID50, lethal dose and infectious dose 3. environment- public health
Keq Range
10^-3
Anticodon
3 nucleotide by which a tRNA recognizes an mRNA codon
What is a System?
A cell or organism, if a system is open that means it exchanges energy with the external environment
Transition state
A chemical intermediate that is higher energy and very unstable.
Methotrexate
A chemotherapeutic agent that is a competitive inhibitor. It binds to the active site of Dihydrofolate Reductase (DHFR) which is involved in the biosynthesis of purines and pyramidines.
Plaque assay
A clearing in a bacterial lawn resulting from lysis by phages.
Feedback inhibition
A common means of biochemical regulation, where no structural resemblance to the substrate or product of the enzyme are born by the regulator.
Holoenzyme
A complete enzyme with its cofactor.
Ternary complex
A consequence of a sequential reaction, occurring when a bisubstrate reaction occurs, consisting of the enzyme and both substrate forms.
S1 pocket
A deep, relatively hydrophobic pocket that can fit long, uncharged side chains of residues such as phenylalanine and tryptophan on chymotrypsin. Binding to this pocket with the appropriate side chain positions the adjacent peptide bond into the active site for cleavage.
Substituted enzyme intermediate
A defining feature of a double-displacement reaction where the enzyme is temporarily modified.
Noncompetitive inhibition
A form of inhibition where the inhibitor and the substrate can bind simultaneously to an enzyme at different binding sites. Decreases the overall number of active enzyme molecules rather than diminishing the proportion of enzyme molecules that are bound to substrate. Appears to 'dilute' the enzyme.
Phosphofructokinase-1 (PFK-1)
A glycolytic enzyme that shows allosteric behavior, it catalyzes the third step in glycolysis
Phosphofructokinase-1 (PFK-1)
A glycolytic enzyme, showing allosteric behavior for which Fructose-2,6-bisphosphate is a positive Allosteric modifier. When bound, it allows the enzyme to be active at a lower [S]
Kcat/Km
A measure of catalytic efficiency
Prostoglandin H2 Synthase 1
A membrane associated protein involved in the synthesis of prostaglandin H2 from the fatty acid arachidonic acid. AA must pass through the hydrophobic channel of the synthase, but can be blocked by aspirin binding to the Serine 530 residue in the hydrophobic channel entrance, near the catalytic site.
Lock and key
A model of ES binding in which the active site of the unbound enzyme is complementary in shape to the substrate.
Induced fit
A model of ES binding in which the enzyme changes shape on substrate binding, the active site forms a shape complementary to the substrate only after the substrate has been bound.
Phosphatidylserine
A phospholipid found on the inner leaflet of the plasma membrane. The polar head is made of a serine.
What is a Favorable Process?
A process that increases entropy and releases energy
Ritonavir
A protease inhibitor of HIV protease, is a transition-state analog.
Negative
A reaction can take place spontaneously if deltaG is _________.
Unfavorable Reaction
A reaction that requires the input of energy and that decreases entropy (endergonic)
Uncompetitive inhibition
A substrate-dependent inhibition where the inhibitor binds only to the enzyme-substrate complex. Cannot be overcome by the addition of more substrate.
Gibbs Free energy
A thermodynamic property that is a measure of useful energy, or energy that is capable of doing work.
Metal ion catalysis
A type of catalysis where a metal ion serves as either an electrophilic catalyst, stabilizing a negative charge on a reaction intermediate, or may generate a nucleophile by increasing the acidity of a nearby molecule, or may bind to the substrate, increasing the number of interactions with the enzyme and thus the binding energy.
General Acid-Base Catalysis
A type of catalysis where a molecule other than water plays the role of a proton donor or acceptor.
Covalent catalysis
A type of catalysis where the active site contains a reactive group, usually a powerful nucleophile, that becomes modified covalently in the course of catalysis.
Reversible inhibition
A type of enzyme inhibition where inhibition can be undone. Characterized by rapid dissociation of the enzyme-inhibitor complex.
Transition-state analogs
A type of irreversible inhibitor that are very potent, and appear similar in structure to the transition state of the substrate of the enzyme.
Lipid anchor
A type of lipid modification that permits a protein to become anchored in the phospholipid bilayer with a lipid tail.
Double-displacement (ping-pong) reactions
A type of multiple-substrate reaction wherein which one or more products are released before all substrates bind the enzyme. The defining feature is the existence of a substituted enzyme intermediate, in which the enzyme is temporarily modified.
Sequential reactions
A type of multiple-substrate reaction, wherein which all substrates must bind to the enzyme before any product is released.
Phsophatidylinositol
A type of phospholipid used in signal transduction, with a inositol group.
Phasphatidylethanolamine
A type of phospholipid wherein which the polar head contains an ethanolamine group.
Viral Replication Steps
A. Attachment B. Penetration C. Un-coating D. Synthesis: Replication Protein Synthesis F. Assembly Maturation Packaging Lysis? H. Release uncoating and lysis not always used
How Fast?
AKA enzymes! Enzymes have nothing to do with the first question of whether a reaction will happen or not they have to do with how fast the reaction will happen
What Will Happen?
AKA what direction will the reaction proceed Will it happen or not? free energy change, the favored direction will be the direction that has a negative delta G aka does not require an input of energy
Biological Catalyst
Accelerates forward and reverse reactions (depending on equilibrium) up to 10^16 times, enzymes only work on thermodynamically favored reactions
Biological catalyst
Accelerates forward and reverse reactions, depending on equilibrium.
Sequential Model
Accommodates negative cooperativity, subunits of allosteric enzyme undergo sequential changes in conformation on binding substrate, binding of substrate to one subunit favors conformational change in adjacent subunit Binding of substrate can decrease activity of enzyme ***** Implied that binding of substrate to one subunit does not immediately change all remaining subunits conformational change allows enzyme to be more or less active
Microenvironments
Active sites are unique ________.
Phosphorylation
Addition of a phosphate group (s) by kinases to serine, threonine and tyrosine hydroxyl groups of other proteins Uses ATP as a substrate
Phosphorylation how?
Addition of a phosphate to a protein
Alcohol and Km
Alcohol Dehydrogenase and Aldehyde Dehydrogenase are two enzymes that metabolize alcohol, some people can drink a lot and others cant this is because if thre is a single base substitution in the genetic code of one of the enzymes it affects the way the enzyme metabolizes alcohol, if it leads to a high Km enzyme a lot of build up of the toxic and alcohol dehydrogenase can't work yet so we experience NAUSEA
What is the importance of Algae and Fungi
Alga produces and secretes carbohydrates; fungus provides holdfast (structure) Economic importance Dyes Antimicrobial (Usnea) Litmus Food for herbivores
Concerted Model
All subunits exist as R or R state, substrate binds readily to R state on the active sites of each polypeptide chain, binding of S disrupts T<-->R equilibrium, accounts for sharp increase in Vo in plot V vs [S] BINDING OF ONE SUBSTRATE TO ONE SUBUNIT WILL CAUSE REMAINING SECTIONS TO MOVE TO R STATE
Premises of concerted model
All subunits exist as either T or R Substrate binds more readily to R active sites on each polypeptide chain ---Accounts for sharp increase in Vo in plot of V vs [S]---
Rapid Responses for Adaptation
Allosteric Control and Reversible Covalent Bond Modification
Rapid adaptive response
Allosteric control, Reversible covalent modification
Cooperativity and Metabolism
Almost every enzyme in metabolism is allosteric, positive or negative cooperation
Concerted model
Also called the MWC model, after those who made it
Allosteric Modifiers
Alter the T <--> R equilibrium
Allosteric enzymes
Always catalyze the committed step of metabolic pathways.
Entropy (S)
Amount of disorder
Long Term Response For Adaptation
Amount of enzyme/protein degradation TAKE AWAY: transcription/processing/translation can be very slow so this takes a long period of time protein degradation is fast but it leads to synthesis over again which is not efficient
NOTE
Amount of product formed is the same with or without the enzyme, just affects how fast
Transition State
An "intermediate" structural stage as a reactant is being converted to product
Primase
An RNA polymerase that makes RNA primers from DNA tempate
tosyl-L-phenylalanine chloromethyl ketone (TPCK)
An affinity label for chymotrypsin which binds at the active site and then reacts irreversibly with a Histidine residue at the site.
Doxycycline
An antibiotic that is a noncompetitive inhibitor of a bacterial proteolytic enzyme, collagenase. Prevents growth and reproduction of bacterial and thereby prevents gum disease.
Proteolytic
An enzyme which catalyzes the hydrolysis of a peptide is called a _______.
Apoenzyme
An enzyme without its cofactor.
Diisopropylphosphofluoridate (DIPF)
An example of a group-specific reagent, that inhibits the proteolytic enzyme chymotrypsin. It modifies one of the 28 serine residues, Ser 195. Also revealed a reactive Serine residue on acetylcholinesterase, an enzyme imperative for nerve impulse transmission.
Penicillin
An inhibitor that consists of a thiazolidine ring fused to a beta-lactam ring to which a variable R group is attached by a peptide bond. Can undergo a variety of rearrangements due to the instability of the beta-lactam ring. Acts as a suicide inhibitor.
Irreversible inhibitor
An inhibitor where the enzyme-inhibitor complex dissociates very slowly because the inhibitor has become tightly bound to the enzyme, either covalently or noncovalently.
Transition state
An intermediate structural stage as a reactant is being converted to product
Kcat
Another name for K2
Rate-limiting step
Another word for the committed step in a metabolic pathway
Transition state analogs
Appear very similar in structure to the transition state of an enzyme's substrate, and act to inhibit the activity of an enzyme by having a much higher binding affinity to the active site than the substrate.
Zymogen activation
Applies to only a selected number of enzymes -Digestive enzymes: Chymotrypsin, trypsin -Enzymes involved in blood clotting cascase Generally irreversible -Involves limited proteolytic cleavage Can be stored and only become active in response to physiological stimuli.
Zymogen Activation Notes
Applies to only a selected number of enzymes (digestive, blood clotting) generally irreversible zymogens can be stored and only become active in response to physiological stimulus
ΔG°'
Assumes [H+] = 10^-7, aka this is for the body because the body's pH is around 7 so it's just a little corrected INDICATES: -whether a reaction releases or requires energy -The ratio of products to substrates at equilibrium -POINT OF REFERENCE for comparison of energetics of all cellular reactions (we can compare one reaction to the next under controlled conditions)
Where Can Velocity Be Determined on M-M Plot?
At the beginning where the slope is linear
What is the order of virus life cycle?
Attachment Asorbtion Penetration assembly/Maturation Exit
Gibbs Free Energy (G)
Available energy to "do work" -refers to the differences in chemical bond energy between products and reactants (this is inherent int he bonds of a molecule, independent of an enzyme) -energy change as a system moved from initial state to equilibrium
3 domains (prokaryotes and eukaryotes)
Bacteria (prokaryotes), Archaea (prokaryotes), and Eukarya (eukaryotes)
Which of the three domains of microbes are prokaryotes and which ones are eukaryotes?
Bacteria-P Archae-P Eukarya-E
Aspartate Transcarbamyolase
Balances the two nucleotide pools for nucleic acid biosynthesis Catalyzes first step in the synthesis of purines and primidines With ATP, the ATCase makes this first step happen but CTP (a nucleotide that is made at the end of this) this is a negative modifier to it binds to ATCase and decreases rate of synthesis
Aspartate transcarbamoylase
Balances the two nucleotide pools for nucleic acid biosynthesis, stimulation by purine ATP nucleotide favors the R state, feedback inhibition by pyrimidine nucleotide CTP favors the T state.
Define nonsense mutation
Base substitution results in a nonsense (stop) codon
Define missense
Base substitution results in change in an amino acid
Coupled Reaction 1 (Krebs Cycle Reactions)
Basically the first reaction has a very high positive ΔG so we would think that it doesn't happen but because of the next reaction having a negative ΔG and using up the oxaloacetate out of the product side of the reaction this means that the reaction will happen because of the reactants to products ratio
Why Do Enzymes Have a pH Optima?
Because certain interactions occur between functional groups of the amino acids in the protein structure and the substrate so for example if an enzyme needs to be de-protonated to have its pH optima and its pKa is 4 it will start working more after the Ph surpasses the pKa it will work best because this side chain will be available to bind/have a negative charge/ etc PROTONATION/DE-PROTONATION OF AMINO ACID DETERMINES PH OPTIMA
Why is Glycine's Kcat/Km Low?
Because not too many contacts in the site so this binding energy is low, strength of enzyme-substrate interactions is not high
How are Enzymes Regulated??
CONCENTRATION, ZYMOGEN ACTIVATION
What Type of Reactions are the Basis of Metabolism?
COUPLED REACTIONS
Negative Feedback on ATCase
CTP is a pyrimidine nucleotide made that is a negative modifier of ATCase so when it binds there is a right shift and the T state is favored so there is a decrease in the rate of synthesis
Fluid Mosaic Model Membrane Dynamics
Can laterally shift or flip flop, laterally shifting is quick but flip flopping is very slow FRAP proves that there is movement (bc there is recovery after the bleaching)I
Viruses
Capsid / Spike / Envelope / Icosahedra, Plaques assay/ Un-coating/ Nuclei Acid
Esterification
Carboxylic acid of fatty acid is esterfied to hydroxyl group of glycerol VERY STRONG COVALENT BOND HOW WE STORE FAT IN THE BODY
Diphosphatidylglycerol
Cardiolipin, a phospholipid present in the membrane of mitochondria. It is two nonpolar tails facing outward from a single polar group.
Mechanisms of Enzyme Regulation
Catalytic activity of an enzyme is regulated so that the amount of product is sufficient to meet the needs of the cell Enzymes can exhibit a range of activity dependent on the physiological state of the cell (example of adaptation)
Carbonic anhydrase
Catalyzes a reaction to turnover 1.0*10^6 molecules/second
Eukarya
Cell Type: Eukaryotic Cell Wall:Varies in composition; contains in carbohydrates Membrane lipids: composed of straight carbon chains attached to glycerol by ester linkage First Amino Acid in Protein Synthesis: Methionine Antibiotic Sensitivity: No rRNA Loop: Lacking Common Arm of tRNA: present
Bacteria
Cell Type: prokaryotic Cell Wall: contains petidoglycan Membrane lipids: composed of straight carbon chains attached to glycerol by esterr linkage First Amino Acid in Protein Synthesis: Formylmethione Antibiotic Sensitivity: yes rRNA Loop: present Common Arm of tRNA: present
Archae
Cell Type: prokaryotic Cell Wall:Varies in composition; contains no petidoglycan Membrane lipids: composed of branched carbon chains attached to glycerol by ether linkage First Amino Acid in Protein Synthesis: Methionine Antibiotic Sensitivity: No rRNA Loop: Lacking Common Arm of tRNA: Lacking
Carbohydrate Addition
Cellular GPS device, self recognition, proteins know where to go, tagging basically O-glycosylation, N-glycosylation GPI anchor
Persistent Viral Infections Examples(Cogenital)
Cervical Cancer(increase cell growth)> Human papillomavirus HIV/AIDS( decrease CD4+T cells)> HIV1 and 2(Lentivirus) Liver Cancer(increase cell growth)>Hepatitis B Progressive Encephalitis(Rapid Mental Deterioration)> Rubella virus
Increase, decrease
Cholesterol can ______ or ________ membrane fluidity, depending on its location.
Cholesterol Effects on the Membrane Phase Transition
Cholesterol does not change the Tm but it minimizes the sharpness of the transition MORE REGULATED
Minimizes sharpness of transition
Cholesterol's effect on the Phase transition.
Covalent modification
Chymotrypsin is a good example of the use of _____________ as a catalytic strategy. The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate, which then becomes covalently attached to the substrate briefly in the course of catalysis.
Zn2+
Cofactor of Carbonic Anhydrase
Latent Viral Infections examples (Cogenital)
Cold Sore(skin and mucous mebrane lesions, genital lesions> Herpes simplex 1 and 2 Leukemia(increase WBC growth)> HTLV_1 and 2 Shingles(Skin lesions)>Varicellovirus(Herpesvirus)
The three types of reversible inhibition
Competitive inhibition, uncompetitive inhibition and noncompetitive inhibition.
Competitive Binding
Competitive inhibitors have similar structure and non-covalent interactions to the substrate so they bind in place of it at the active site
Reversible Inhibitors
Competitive, uncompetitive, noncompetitive
Phospholipids
Comprised of a Glycerol backbone Fatty acids at position 1 and 2 hydroxyl groups Polar group at position 3 hydroxyl group - Typically something like phosphate then alcohol
Cholesterol
Comprised of a steroid nucleus with a key hydroxyl group giving this molecule aphipathicity. Not found in plants, only animals.
Models to Explain Allosteric Behavior
Concerted model or sequential model
ES <---> EP
Conversion of bound substrate to bound product
Irreversible Inhibitors
Covalently bond to enzyme or non-covalently bond to enzyme and bind so tightly that they do not come off (very slow dissociation)
prokaryotic cells
DNA: One circular; some two circular; some linear Histones: In archae First Amino Acid in Protein Synthesis: Formylmethionine(bacteria) Methionine (archae) Ribosomes: 70S Growth: Binary Fission
Eukaryotic Organelles
DNA: circular Histones: No First Amino Acid in Protein Synthesis: Formylmethionine Ribosomes: 70S Growth: Binary Fission
Eukaryotic Cells (Mitochondria and Chloroplast)
DNA: linear Histones: yes First Amino Acid in Protein Synthesis: Methionine Ribosomes: 80S Growth: Mitosis
How Do Enzymes Increase Reaction Rate
Decrease Activation Energy
Biological catalyst
Decrease the activation energy in a biological system for a chemical reaction
Membrane fluidity
Dependent on properties of fatty acid chains in phospholipids
Chemical Reaction Itself
Depends on the actual reaction and what we are trying to break and form, it is different for different molecules depending on polarity, etc
Whether a Reaction Happens at All
Depends on the free energy difference (delta G) between products and reactants, determines whether a reaction will occur spontaneously
First step in determining the chemical mechanism of an enzyme
Determination of which functional groups are required for enzyme activity.
Lipid addition
Determine the intracellular location and activity of an enzyme
Carbohydrate addition
Determine the intracellular/extracellular location of the enzyme that is modified by this.
Specificity
Determined by the number and type of noncovalent interactions
Unstable
Difficult to isolate, this is what distinguishes it from a true reaction intermediate, Instability due to the strain on covalent bonds of the original reactant (this has to happen in order to convert reactant to product
Thermodynamics Predicts the ________ of a Reaction
Direction
No, they only shift how fast equilibrium is achieved, not the concentrations at equilibrium.
Do enzymes alter the equilibrium constant? Why?
Allosteric Enzymes
Do not show MM kinetics, they are sigmoidal showing cooperativity, MM is used in labs for drug inhibition usually
Biological catalyst
Doesn't alter dG^o' or Keq in a biological system. It does accelerate the attainment of equilibrium, but does not change where the equilibrium falls.
Unsaturated
Double bond, more spread out, more fluid/permeable
Internal energy
E - a function that keeps track of heat transfer and work expenditure in the system
Enzyme-Substrate Complex
ES
What is tissue culture/cell culture/monolayer? What is their use?
In cell cultures Tissues are treated with enzymes to separate cells Virally infected cells are detected via their deterioration, known as the cytopathic effect (CPE) Continuous cell lines are used Monolayer-cells stuck at bottom Normal cell has monolayer, Transforme/cancer cell doesn't Normal and transformed ar combine by hydrobrima
Keq
Indicates the tendency of a reaction to go toward completion.
Example of Operon
Inducible genes are lactose operons o They are usually off unless there is a substrate that needs to be broken down/sugar to metabolize (sugar catabolism) o If lactose is available repressor binds to the metabolite of lactose and lactose-digesting enzymes are created Repressible Genes are Tryptophan Operon o When lactose is not available a repressor binds to the operator site and prevents transcription • Repressible operons are always on o Synthesis of proteins always needs to occur unless an amino acid comes from outside the (anabolism) cell o Structural genes are transcribed unless turned off
Uncompetitive Binding Graph
Inhibitor binds to ES complex making the complex very stable and lowering the Km but it also takes a bunch of enzymes out of business so it plateaus and has a lower Vmax
PhosphatidylSERINE
Inner leaflet in PM
Bioenergetics
Interactions and reactions lead to energy transformations
Palmitoylation
Internal SH of cysteine targeted with the modification of a 16 carbon fatty acid
Bacteria
Intracellular Parasite: No Plasma Membrane: Yes Binary Fission:Yes Pass through Bacteriological Filters:No Posses Both DNA and RNA: Yes ATP generating Metabolism: Yes Ribosome: Yes Sensitive to Antibiotics:Yes Sensitive to interferon:No
Virus
Intracellular Parasite: Yes Plasma Membrane: No Binary Fission:No Pass through Bacteriological Filters:Yes Posses Both DNA and RNA: No ATP generating Metabolism: No Ribosome: No Sensitive to Antibiotics:No Sensitive to interferon:Yes
Rickettsia / Chalmydia
Intracellular Parasite: Yes Plasma Membrane: Yes Binary Fission:Yes Pass through Bacteriological Filters:No/YES Posses Both DNA and RNA: Yes ATP generating Metabolism: Yes/NO Ribosome: Yes Sensitive to Antibiotics:Yes Sensitive to interferon:No
Zymogen Activation Definition
Irreversible conversion of an inactive precursor to active enzyme by cleavage of covalent bonds, advantages because dangerous enzymes we don't want them active all the time, disulfide bonds keep it all as one unit
Affinity labels (substrate analogs)
Irreversible inhibitor molecules that covalently modify active-site residues and are structurally similar to the enzyme's substrate. Very specific, more so than group-specific reagents.
Suicide inhibitors (mechanism-based inhibitors)
Irreversible inhibitors that are chemically modified substrates, which binds normally and initiates catalytic mechanisms, but which form a reactive intermediate that inactivates the enzyme through covalent modification. Is used by researchers, as it provides the most specific and selective means of modifying the active site.
Specificity
Is controlled by structure
Km
Is equal to the substrate concentration at which the reaction velocity is half its maximal value.
Important Question in Thermodynamics
Is this process favorable? ... we mean does it increase entropy and release energy
Main Differentiation with ΔG°'
It is a point of reference for the cells, the body cannot have this actually but it's basically like the closest we can get in the lab to the cellular standards so our actual measured in our body is just ΔG and we compare it to this
Concentrations of the reactants and products
It is important to stress that whether the dG for a reaction is larger, smaller or the same as dG^o' depends on the _______________.
Allosteric Behavior and Regulatory Enzymes
It's the cells' way to respond to environmental signals, it is an adaptive response
Vo
K(2)[ES]
Vo Eq.
K2 [ES]
Vmax Eq.
K2 [E]T
K1
Kcat/Km is set by, the rate of formation of the ES complex
Vo
Kcat/Km*[S]*[E]t, when [S] << Km
Why is Activation Energy Crucial?
Keeps molecules from falling apart, it is like a little hump of protection making it so there has to be an increase in energy to make the bonds break apart, if there was no activation energy all molecules would just fall apart
ΔG°' = 0
Keq=1, the reaction is at equilibrium
Lytic Cycle
Kills host a mechanism of phage multiplication that results in host cell lysis
How fast
Kinetics predicts ______ ______ a chemical reaction will occur.
Km Equation
Km = (K-1) + (K2) / (K1) Basically the disappearence of the ES complex divided by the formation of the ES complex so a large Km means that the ES is unstable and a small Km means that the ES is stable
Competitive Binding Graph
Km is increased so the ES complex is less stable and the binding is weak so for this reason we can still reach the same Vmax it just requires an increased substrate concentration, we just have to have enough substrate available that it binds and converts to product
Hexokinase
Km= .05 mM, in other tissues and takes glucose and phosphorylates it for energy for body cells
Glucokinase
Km= 5mM, is found in the liver and works when the glucose levels are high and then phosphorylates this glucose to be stored as glycogen
Increase in Non-Covalent Interactions....
Leads to an increased likelihood that the substrate will be converted to product
Small Change in Allosteric Modifiers
Leads to big changes in enzyme activity
PhosphatidylinOSITOL
Least common, signal transduction
Membrane Fluidity
Length of chain Double bonds/packing
Visual of a Favorable Reaction
Like a ball rolling down a hill this is favorable because higher energy at top and lower at the bottom so energy is released (exergonic) AKA reactants at top have more energy than the products at the bottom
Advantages of low Km
Limited [S] Solubility Osmotic effects
Solution to Problem with MM
Linear transformation of the equation -Lineweaver-Burk Plot -Hanes-Woolf Plot
Location Modifications
Lipid addition, carbohydrate addition
Active site
Location where substrate binds to the enzyme (also where the enzyme binds a cofactor, if applicable)
Binding of Active Site Can Occur As...
Lock and key OR induced fit
Instability Leads to
MAXIMAL BINDING ENERGY RELEASE OF THIS ENERGY
Specificity Results From...
MAXIMIZED BINDING CAPACITY
Phospholipids
Make up membrane Glycerol backbone fatty acids polar group
Why is this Important?
Makes it so cells only make what they need, no more than what is absolutely necessary
Kcat/Km
Measure of catalytic efficiency Most enzymes in the cell are not saturated with substrate, considers rate of catalysis with a particular substrate, ***strength of enzyme-substrate interactions
Km
Michaelis constant, related to rate constants of individual steps in catalysis, also a measure of the strength of the ES complex RATIO OF RATE CONSTANTS PREDICTS STABILITY OF ES COMPLEX
DiphosphatidylGLYCEROL
Mitochondria!!!
dG^o'
Modified standard state. Indicates whether a reaction releases or requires energy. The amount of energy involved. The ratio of products to substrate at equilibrium. The point of reference for comparison of energetics of all cellular reaction.s
Turnover Number (Kcat)
Moles of substrate converted to product per mole of enzyme (at saturation) per second Vmax/[ET] AKA how fast can an enzyme convert substrate to product per unit time when it's saturated
PhosphatidylCHOLINE
Most common in PM
Phosphorylation
Most important covalent modification Kinases add phosphates to OH groups of (serine/threonine/tyrosine), USES ATP AS SUBSTRATE phospotases remove them can be very specific for the proteins they modify more general, used in metabolism a lot
List features of Mycoplasma
Mycoplasmatales -Lack a cell wall; pleomorphic -M. pneumoniae causes mild pneumonia
Myristoylation
NH of N-terminal Glycine targeted with a 14 carbon fatty acid addition type of modification.
Can We Use Lineweaver Burk Plots?
NO because the graph to begin with was not hyperbolic
Substrate Bonding, How Must it be Bonded?
NOT TOO STABLE because product will never be formed NOT TOO WEAK it wont stay on enzyme long enough for product to be formed
Integral Proteins
Need harsh conditions to be removed from membrane, hard to study bc must mimic conditions and have to have some hydrophobic characteristics to interact with lipid tails in the middle of the membrane
List some chemical agents that can cause cancer (mutagens) and describe their mechanism of action?
Nitrous acid: causes adenine to bind with cytosine instead of thymine Nucleoside analog: incorporates into DNA in place of a normal base; causes mistakes in base pairing
Trans
No bend, less fluidity
Saturated
No double bond, less fluidity
Noncompetitive Binding
Noncompetitive inhibitors bind to a site on the enzyme that is not the active site and cause a conformational change in the enzyme so that it can make noncovalent interactions with the substrate but not enough necessary to convert it to a product
DNA Vaccine
Nonpathogenic viruses carrying genes for pathogen's antigens
Stop Codon
Nonssence Codon -Don't code for amino acid
Peripheral Proteins
Not strongly bound, they can be dissociated with mild detergent treatment or with salt concentrations
Six
Number of enzyme groups (classification)
Rate of Catalysis
Number of moles formed per unit of time
Two
Number of phases of the reaction of chymotrypsin
Cholesterol Decrease Fluidity
OH groups form H bonds with carbonyl of phospholipid head group, can form complex with SPHINGOLIPIDS, may increase rigidity and order regions of the membrane (LIPID RAFTS)
Gastroesophageal Reflux Disease (GERD)
OMEPRAZOLE TREATS IT, acid content goes up into esophagus and this is because the esophageal sphincter valve is not closing properly so as a result some of the acid goes up into the esophagus which isn't protected by mucus lining
Binding must be...
OPTIMAL (happy medium)
Omeprazole
OTC drug that needs to be metabolized by one of the cytochrome P450 members and there's a genetic polymorphism in this Sulfur double bonded to oxygen, the bond gives you 2 SH groups turning sulferic acid to sulfenamid giving a SN bond and sulfenamide binds covalently to proton and K and ATPase to help maintain acid environment in stomach
Some extra explanation
Omeprazole has sulfur double bonded to oxygen and when it goes into the stomach this is a very acidic environment so the protons take over and make the group SH and this H leaves and the S is able to bond to the proton pump in the cell and it is an inhibitor so it decreases the activity of this pump making less protons pumped out, therefore regulating the acidity in the stomach that is the problem
RNA Polymerase
Only in RNA Copies RNA from DNA template
1-2
Optimal pH of Pepsin? Range.
7-9
Optimal pH of chymotrypsin? Range.
Ectotherms
Organisms that assume the temperature of the environment
Endotherms
Organisms that maintain a constant body temperature
Acetylation
Other covalent modification We add an acetyl group (no charge) to loosen electrostatic interactions binding to N terminus
ADP Ribosylation
Other covalent modification significant because introducing potential steric constraints/charges/hydrophobicity when this happens it drastically alters the activity of enzmye
dG
Overall free energy change of the reaction
which of the three domains of microbes are prokaryotes and which ones are eukaryotes
PRO: archaea and bacteria EUK: eukaryotes
Types of Irreversible Inhibitors
Penicillin, aspirin, GERD example
Specificity Example
Peptide bond cleavage by proteolytic enzymes
Which AA adjacent to peptide bond will chymotrypsin cleave?
Phenylalanine because it has high catalytic efficiency
Amphipathic
Phospholipids are ______ molecules - they have both hydrophilic and hydrophobic characteristics
Amphipathic
Phospholipids are amphipathic molecules, they have both hydrophilic and hydrophobic characteristics
Reversible covalent modification
Phosphorylation Acetylation ADP-ribosylation
Rapidly
Phosphorylation/dephosphorylation _______ alters the activity of an enzyme
Phase Transition (Tm)
Point where goes from solid to liquid, temperature affects fluidity of membrane, melting temp depends on FA chain length and extent of unsaturation EX. PCR
Name different shapes of viruses. Which one is unique to viruses?
Polyhedral -Capsid -Capsomere -Nucleic Acid -many faces Helical -Capsid -Capsomere -spiral shape -Nucleic Acid Complex -Nucleic Acid -Tail -Sheath Ex: T virus, bateriophage Enveloped helical -envelope -Nucleic Acid -capsomere -spike Ex:Flu virus
Enzymes are Stereospecific
Position matters, non-covalent interactions stabilize substrate binding,
Zero-order kinetics
Predication - when [S] >> Km V = Vmax Enzyme is saturated V is independent of [S]
First-order kinetics
Prediction - when [S] << Km V is proportional to [S]
Kinetics
Predicts how fast a reaction will occur.
Thermodynamics
Predicts the direction of a chemical reaction.
Where in eukaryotes one can find DNA?
Prokaryotes -There are no membrane bound organelles, so DNA is found free floating in the cytoplasm -In the nucleoid region -Found in a coiled loop -DNA can be in the form of plasmids in certain bacteria; distinct from the chromosomal DNA
Allosteric Behavior and Rate Limiting Step
Rate limiting steps are when the molecule/substrate cannot be used in any other way it has no choice but to continue on to a certain pathway, this step is so important which is why allosteric enzymes regulate it HIGHLY REGULATED
Group Specific
React with R groups of amino acids, SPECIFICITY
Catalysis
Reaction rates are increased by enzymes to meet the demands of a cell (LOWERS ACTIVATION ENERGY)
Endergonic
Reaction where energy input is required to occur
Exergonic
Reaction where energy is released
First-order reactions
Reactions in which the velocity is directly proportional to the reactant concentrations
Second-order reaction
Reactions that are bimolecular, they include two reactants
Pseudo-first-order reactions
Reactions which are second-order but appear first-order due to the fact that the concentration of reactant B is much greater than the concentration for that of A, which causes the rate of the reaction to appear to depend solely on B.
Lipid raft
Regions of the phospholipid bilayer made very rigid (low fluidity) by the presence of cholesterol in certain orientations.
Features of allosteric enzymes
Regulation of catalytic activity by environmental features, including the final product of the metabolic pathway regulated by the enzyme; kinetics that are more complex than those of Michaelis-Menten enzymes; quaternary structure with multiple active sites in each enzyme.
MM Equation
Relates initial and maximum rates of enzymatic reaction AT ANY [S] (basically the maximum velocity for that reaction at that time)
EP <---> E + P
Release of product
Protoplast fusion
Removing cell walls from two bacteria allows them to fuse
Replication
Replication o One strand of DNA serves as a template for the production of a second strand o The strand is opened up by topoisomerase and gyrase- these enzymes relax the strand and unwind the double helix and supercoiling o The double strand of DNA are separated by Helicase- enzyme breaks the hydrogen bonds between the two strands of DNA o A replication fork is created o DNA polymerase adds new nucleotides in the 5' to 3' direction,synthesizes DNA and proof reads/ repairs the DNA o The leading strand (5'-3') is synthesized continuously o The lagging strand (3'-5') is synthesized discontinuously through Okazaki fragments o Primers are needed to initiate synthesis for both strands o The Okazaki fragments are joined together by ligase o DNA ligase- makes covalent bonds to join DNA strands Topoisomerase- relaxes the super coiling ahead of the replication fork and separated DNA circles at the end of replication
5.What are the products of: Replication, Transcription, Translation and Reverse Transcription?
Replication: DNA Transcription: mRNA Translation: Proteins Reverse Transcription:DNA/ parts of a desired gene , DNA copy of mRNA is produced
What is meant by obligate intracellular pathogens?
Require living host cells to multiply
Problem with MM
Requires extrapolation of asymptotes (have to estimate the Vmax)
Catalytic groups
Residues of the enzyme which directly participate in the formation and breakage of bonds.
Types of Inhibitors
Reversible, Irreversible
Allosteric control
Role of allosteric modifiers Feedback inhibition in metabolic pathways
Allosteric Control
Role of allosteric modifiers, feedback inhibition in metabolic pathways
scientific and agricultural applications of recombinant DNA
SCIENCE -vaccines: safer -protein synthesis -genetic screening: look for diseases from mutations, identify pathogen DNA in blood/tissue -gene therapy: replace damaged genes, good in treating CF, sickle cell, hemophilia, diabetes -xenotransplants: grow organs in animals and put in humans -producing insulin AGRICULTURE -herbicide resistence -salt tolerance -freeze resistance -pest resistance -improving nutritional value of crops
Prenylation
SH of cysteine targeted with this type of modification. Not a fatty acid addition 5 Carbon repeating units Can be used in cholesterol metabolism Can also anchor
What is Specificity Controlled By?
STRUCTURE, determined by the type and number of noncovalent interactions
Describe the role of Yeast cells in making gene products
Saccharomyces cerevisiae -Easily grown and has a larger genome than bacteria -Expresses eukaryotic genes easily( human or plant cells)
Cis
Same, bend in it, causing more fluidity/permeability
Trans
Saturated fatty acid conformation
Positive Modifiers
Shift curve left, more hyperbolic, achieving same philosophy with not as much substrate, positive allosteric modifier is bound (enzymes work over a range of [S])
Aspirin
Short lived, has an acetyl group that inhibits prostaglandin synthase, PROSTAGLANDINS associated with inflammation and we need this synthase to make prostaglandin so this is why when we take aspirin it brings down inflammation Synthases have a hydrophobic pocket because the substrate is a long fatty acid, serine at the edge of the pocket and aspirin's acetyl group covalently bonds with this OH group and through steric effects that acid cannot bind the way that it needs
Michaelis-Menten Plot
Shows RELATIONSHIP between substrate concentration and reaction rate (velocity) Concentration if on the x axis and velocity is on the y axis
Increase in Activation Energy...
Slower Reaction
Gene Silencig
Small interfering RNAs (siRNAs) bind to mRNA, which is then destroyed by RNA-induced silencing complex (RISC)
Cofactors
Small molecules which enzymes rely on the presence of to function properly.
Coenzymes
Small organic molecules derived from vitamins
Micro-Environment
Smaller environment within a larger one, we want to get molecules in a confined environment so the chances of them bumping into each other is much greater (the active site is this micro environment)
General properties of enzymes
Specificity Catalysis Regulation
General Properties of Enzymes
Specificity, catalysis and REGULATION
Positive Effectors
Stabilize R state, higher affinity, higher rate of formation, R state more active state
Negative Effectors
Stabilize T state, lower affinity, lower rate of formation, T state less active state
genetics
Study of genes, how they carry information, how information is expressed, how genes are replicated
Suicide Inhibitors
Substrate analogue leads to enzyme inactivation, react with side chains of AAs in active site
Lock & Key
Substrate binding to enzyme is a perfect, complementary fit
Induced Fit
Substrate binds to enzyme, does not fit perfectly but causes a noticeable conformational change that leads to different conformational state
BINDING TOO WEAK
Substrate will be moving on and off the enzyme too quickly so it will not stay on long enough to be converted to product
BINDING TOO TIGHT
Substrate will never get to transition state
Weak attractions
Substrates are bound to enzymes by multiple ____________.
Sequential model
Subunits of allosteric enzyme undergo step by step changes in conformation on binding substrate. Binding of substrate to one subunit favors conformational changes in the adjacent subunit ---Accommodates negative cooperativity---
Aspirin (acetylsalicylic acid)
Suicide inhibitor of prostaglandin H2 synthase 1.
Penicillin
Suicide inhibitor of the enzyme involved in bacterial wall synthesis, the peptide bond in beta-lactam ring of penicillin mimics the transition state complex (PENICILLIN IS A TRANSITION STATE ANALOGUE) Reacts with side chains of active sites and stays here as a big bulky molecule making the enzyme not work When penicillin binds the bacteria are unable to make a cell wall (affects gram positive bacteria because gram negative do not have a cell wall)
Second Law of Thermodynamics
Systems tend to proceed from ordered to disordered states and all processes proceed toward equilibrium (minimum potential energy)
The second law of thermodynamics
Systems tend toward disorder and randomness
congenital infections caused by viruses
TORCH toxoplasma other rubella cytomegalovirus (CMV) herpes parvovirus varicella enteroviruses
trophozoite vs cyst
TROPH -feeding and growing stage of protozoa -cannot survive out of host -disease causing state -divides via binary fission -labile cell membrane CYST -non dividing -rigid cell host -can survive outside of cell host -transmission state -resistant to H2O and dessication
Enhances
Temperature ______ the rate of enzyme-catalyzed reactions, the a point.
An assumption of ensemble studies
That all enzymes present in the mixture that is being studies are the same or very similar.
His 57 (Histidine 57)/(57)
The Histidine present in the active site of chymotrypsin.
Lower
The Km-app of an enzyme after the addition of an uncompetitive inhibitor will be _____ to the Km it would be otherwise, due to the lower concnetration of S required to form half of the maximal concentration of ES.
Noncompetitive Binding Graph
The Vmax is decreased, the inhibitor lowers the concentration of functional enzymes, so binding is happening but not converting to product whereas in competitive the inhibitor binds and then comes off so the opportunity is there to work
Lower
The Vmax of an enzyme after the addition of an uncompetitive inhibitor will be _____ to that of the Vmax without any inhibitor.
Fluid Mosaic
The _____ ______ Model describes membrane dynamics
delta G
The ________ of a reaction depends only on the nature of the reactants and on their concentrations
specificity
The _________ of binding depends on the precisely defined arrangement of atoms in an active site.
Catalysis Def
The acceleration of a chemical reaction by a catalyst
Relaxed (R)
The active conformation, which catalyzes reactions
small
The active site takes up a ____ part of the total volume of the enzyme.
Km
The activity of allosteric enzymes is more sensitive to changes in substrate concentration near ______ than are Michaelis-Menten enzymes with the same Vmax.
Enzyme Size and Allosteric Behavior
The behavior could describe why they are larger, because they need a bunch of sites to be modified in different ways there isn't just an active site
R
The binding of substrate disrupts the T<->R equilibrium in favor of _____.
Hydrolysis of ATP
The breakdown of ATP to release energy to be used by the cells, this is favored and conversely the formation of ATP requires energy
Esterified
The carboxylic acid of a fatty acid is _______ to the hydroxyl group of glycerol
The Change in Free Energy for a Reaction is Equal to...
The change in bond energy minus the temperature multiplied by the change in entropy
delta H (dH)
The change in enthalpy described in the Gibbs free energy equation. A measure of the overall change in bond energy when bonds are broken or formed in a reaction. Is a measure of the distance from the equilibrium of a reaction.
So basically ΔE is...
The change in internal energy is the sum of the heat absorbed by the system and the work done on the system
N-acetyl-L-phenylalanine p-nitrophenyl ester
The chromogenic substrate for chymotrypsin, used to easily monitor chymotrypsin in a laboratory by the color formed. It is a substrate analog.
The Catalytic triad
The constellation of residues present in the active site of chymotrypsin.
Energy-producing, energy-requiring, metabolism
The coupling of ______-______ and _____-______ reactions is a central theme in the ___________ of all organisms.
ES complex
The critical feature of the Michaelis-Menten's treatment is that a ________ is a necessary intermediate in catalysis.
Free energy of the products
The delta G of a reaction depends only on the _____________. (Path does not matter, only the goal)
Km
The determined characteristic of an enzyme found with an ensemble study, others can be determined as well.
Gout
The disease caused by excess urate crystallization in the fluid and lining of the joints. Can be caused by the loss of function of an enzyme, PRS which catalyzes PRPP which is a vital precursor for all nucleotide synthesis, purine formation in particular.
Heterotropic effects
The effects of regulatory molecules on allosteric enzymes
Binding energy
The energy released on binding. Free energy is released by the formation of a large number of weak interactions between a complementary enzyme and substrate.
Activation Energy (ΔG‡)
The energy required to reach the transition state What stands between substrate and product
Stabilization of the transition state
The essence of catalysis is _______________.
dG^o
The free energy change under standard states
Activation Energy (ΔG‡)
The free energy necessary to initiate the conversion of reactants to products, ΔG tells us whether it is forward or reverse but it does not tell us the energy required to initiate the conversion (SEPARATE TERM AKA ACTIVATION ENERGY) The energy required to reach the transition state What stands between substrate and product
Binding Energy
The free energy released when the multiple non-covalent interactions are formed as the substrate binds to the enzyme
In the transition state
The full complement of weak interactions is formed only when the substrate is ________. (some sort of state)
Competitive inhibition
The hallmark of _________ is that it can be overcome by sufficiently high concentrations of substrate.
Vo
The initial rate of catalysis, the number of moles of product formed per second when the reaction is just beginning. Used to simplify reaction schematics to ignore reverse reactions.
Tense (T)
The less active/inactive conformation of an allosteric enzyme. Is more stable, and therefore in greater concentration when in the lack of substrate or signal molecules
Delta G (dG)
The magnitude of _____ depends on: 1) The chemical reaction itself 2) How far the system is from equilibrium 3) [Reactants] and [Products] and number of bonds broken and made
Posphatidylcholine
The most common phospholipid found in the plasma membrane.
Apparent Km (Km-app)
The new value of Km for enzymes being observed with competitive inhibitors, due to the change in concentration required to obtain the same Vmax.
Going from E + S to ES, what is important here?
The number and strength of noncovalent interactions are important here because if there is TOO FEW/NOT STRONG ENOUGH ES does not stay and it goes back to E + S, we want to favor formation of ES because we want product
Turnover number
The number of substrate molecules that an enzyme can convert into product per unit time when the enzyme is fully saturated, determined by Vmax.
Symmetry rule
The premise of allosteric enzymes that require that there are no hybrid R/T state enzymes.
Transformation
The process in which gene are transferred from one bacterium to another as naked DNA -pick DNA from outside(enviroment) and then incorporated into chromosome 1. Recipient cell takes up donor DNA 2. Donor DNA aligns with complimentary bases 3. Recombination occurs between donor DNA and recipient DNA
Burst phase
The quicker/more reactive phase of the chymotrypsin reaction
Kd
The rate constant for the dissociation of the ES complex, also called K1
K-1
The rate constant for the dissociation of the ES complex, also called Kd
K2
The rate constant for the formation of product P
K1
The rate constant for the formation of the enzyme-substrate complex
K-2
The rate constant for the reaction going from P to the ES complex, in that direction.
Negative
The reaction is spontaneous if dG is ______.
High Keq
The reaction will proceed until conversion of reactants to products is nearly complete
Ser 195 (Serine 195)/(195)
The reactive serine residue in Chymotrypsin
Equilibrium
The regulatory molecules alter the ________ between T and R forms.
Ways allosteric enzymes are distinguished
The response to changes in substrate concentration and to the susceptibility to regulation by other molecules.
Gel electrophoresis
The separation of substance( such as proteins or DNA) by their rate of movement through an electrical field
Uncoating
The seperation of viral nucleic acid from its protein coat
Three-dimensional cleft or crevice
The shape of the active site, formed by groups that come from different parts of the amino acid sequence.
Oxyanion hole
The site that stabilizes the inherently unstable first tetrahedral-intermediate's negative charge on the oxygen which was the carboxyl group. Made of a Glycine (Gly 193), whose NH groups help stabilize the negative charge by hydrogen bonding.
Committed step
The step taken in an enzymatic pathway, such as A -> B, where subsequent steps are required to reach product, and A -> B has no other options after occurrence other than formation of product.
In singulo
The study of a single enzyme at a time in place of the study of many enzymes at a time (ensemble).
Ensemble Studies
The study of all of the enzymes present in a solution
Kinetics
The study of the rates of chemical reactions
Enzyme kinetics
The study of the rates of enzyme-catalyzed reactions
Sigmoidal curve
The substrate vs reaction velocity graph of an allosteric enzyme has this shape.
Why is Equilibrium a Problem?
The systems will always go toward equilibrium but this is a problem because there is no free energy for the cell to do work at equilibrium NO ENERGY TO DO WORK HERE
Aspartate 102 (Asp 102)(102)
The third and final residue involved in catalysis in the active site of chymotrypsin, which hydrogen bonds to the NH of the imidazole ring.
The first law of thermodynamics
The total energy of a system is conserved
Covalent
The type of bond an ester bond is
Ordered
The type of sequential reaction wherein which the substrates bind the enzyme in a defined sequence
Random
The type of sequential reaction wherein which the substrates bind the enzyme in no defined sequence.
Lower, unchanged
The value of Vmax of an enzyme after a noncompetitive inhibitor is added will be _____ than it would be otherwise, whereas the Km will be _____.
Vmax
Theoretical maximal rate of reaction but is NEVER achieved in reality, to reach Vmax would require that ALL enzyme molecules are tightly bound with substrate, Vmax is asymptotically approached as substrate is increased
Similarities of Membranes
There are differences in membranes but all have a bilayer and polar headgroups interact with water and nonpolar dont, hydrophobic effect organizes it
System is at equilibrium
There is no net change in the concentrations of the products and reactants and the delta G is zero if the ______________.
Low Keq
There is not much product being produced
Direction
Thermodynamics predicts the _______ of a chemical reaction
Hydrolyases
These enzymes cleave molecules by the addition of water. Ex. Trypsin
Ligases
These enzymes join two molecules in a reaction powered by ATP hydrolysis.
Isomerases
These enzymes move functional groups within a molecule.
Oxidoreductases
These enzymes transfer electrons between molecules, catalyzing oxidation-reduction reactions. Ex. Lactate dehydrogenase, glucose degradation
Transferases
These enzymes transfer functional groups between molecules.
Irreversible inhibitors
These inhibitors that bind covalently to an enzyme can be used for elucidating the mechanisms of enzymes.
Group-specific reagents
These irreversible inhibitors modify R groups of amino acids.
Peripheral proteins
These proteins are mainly surface level or attached to other enzymes protruding from the membrane.
Lipid anchored proteins
These proteins are peripheral but are anchored into the phospholipid bilayer using an anchor lipid which is embedded with the polar tails of the bilayer.
Peripheral proteins
These proteins can be removed by adding high concentrations of salt or very mild detergents
Where Do Substrates Bind/What are they
They are physiological enzymes that bind to allosteric site (different site than active site) this leads to rapid changes in enzyme activity THIS IS NOT INHIBITION, do not mix up
Proteolytic Enzymes
They cleave peptide bonds between amino acids, TRYPSIN cleaves when LYS or ARG are attached to the carbon next to the bond, THROMBIN only will cleave if it is ARG
Penicillin
This antibiotic is an important irreversible inhibitor which inhibits the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls and killing the bacteria.
Transition State High Free Energy
This is because the bonds are strained so the substrate is wanting to increase the entropy and disorder almost breaking apart and having so much ability to do work because not in bonds ENZYME STABILIZES THIS normally a lot of energy would have to be put in to make the molecules form a structure but because they are stabilized by the enzyme and not completely disordered less energy is needed to create the bonds and product product
ΔG‡
This is the ACTIVATION ENERGY, determines the rate of reaction
ΔG°' is Positive
This means that energy is required to make the reaction happen forward, it is going to happen in the reverse and there is going to be less products than reactants causing a larger denominator and a smaller Keq
Low Km
Tight binding between E and S, enzyme functions at low concentrations [S] ~ 10^-9 M
Low Km
Tight binding, [S] around 10^-9 M
Prosthetic (helper) groups
Tightly bound coenzymes
Phase transition
Tm, temperature affects the fluidity of the membrane
Aminotransferases
Transfer amine groups between donor and acceptor molecules. Used in amino acid synthesis and degradation.
Main Enzyme Example
When making carbonic acid we need carbonic anhydrase because the rate enhancement is 7.7x10^6, the same equilibrium would be reached but it happens in seconds with the enzyme instead of hours
Feedback inhibition
Where a large amount of product at the end of an enzymatic pathway/sequence inhibits the enzyme catalyzing the committed step
Active Site
Where substrate binds to an enzyme, active site can contain amino acids from different positions in the linear sequence (this is because they are brought together by folding) Location where substrate binds to the enzyme (also where the enzyme binds a cofactor, if applicable)
Competitive inhibition
Where the inhibitor resembles the substrate and binds to the active site of the enzyme. It diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.
concentration of reactants and products
Whether dG is >, <, or = dG^o' depends on the _______________________________.
ΔG vs. ΔG°'
Whether ΔG is >, < or = ΔG°' depends on the concentration of reactants and products ** reactions that are not spontaneous based on ΔG°' alone can be made spontaneous by adjusting the combination of reactants and products the difference between the two is the basis for coupled reactions
w
Work done ON the system
yeast, plant, and mammalian cells in forming gene products
YEAST -yeast carry plasmids which are transferred to yeast cells whose walls have been removed -yeast will express more foreign genes than bacteria, bc euk -will continuously secrete product MAMMALIAN -growing certain mam cells in cultures as hosts for growing viruses -best suited for protein products for med use bc cells secrete their products and low risk of allergens/toxins -cloning the gene in bacteria and then inserted into mammalian cells PLANT -grown in culture, altered by rDNA techniques, generate genetically modified plants -can make human vaccines and antibodies -use bacterium to do this
What Happens When You Are at a Non-Linear Part of Graph
You will have to perform a dilution to move it down to the concentration on M-M where it is linear rate
Vmax
[E]t*K2
dG
_______ is the relevant term for living systems.
An enzyme
_______________ cannot alter the laws of thermodynamics and consequently cannot alter the equilibrium of a chemical reaction.
base analogs
a molecule that resembles a base pair but does not pair or bond with the match which can induce mutation
mutagens
a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations
Fructose 2,6-biphosphate
a positive allosteric modifier for PFK-1, it allows PFK-1 to be active at a lower [S], makes the graph more hyperbolic MORE FRUCTOSE 1,6 PHOSPHATE CAN BE MADE AT LOWER CONCENTRATIONS OF SUBSTRATE (FRUCTOSE 6-phosphate) WHEN THERE IS FRUCTOSE 2,6 BIPHOSPHATE PRESENT
Codon
a sequence of 3 nucleotide in mRNA that specifies insertion of an amino acid into a polypeptide
The Genetic Code
a set of rules that determine how a nucleotide sequence is converted to an amino acid sequence of a protein mRNA and the aa they encode
epidemic
affects many people at the same time, more than expected
Define base analogs
aka nucleoside analog - incorporates into DNA in place of a normal base; causes mistakes in base pairing -chemical mutagen
double-helix
all cells DNA are in double-stranded form (even plasmid, mitochondria, and chloroplasts), connected through hydrogen bonds, like a twisted ladder
Mutagens
an agent in the environment that bring about mutation
First Law of Thermodynamics
Energy is neither created or destroyed it just changes form
Enzyme and Bonds
Enzyme weakens covalent bonds in the substrate, bc enzyme wants to convert it to product
E + S ---> P + E , what should we take away from this?
Enzymes return to their original conformations, they are catalysts and are not used up during the reaction they remain at the beginning and end and then can be used again
Kinases
Enzymes that add phosphate group(s) to serine, threonine and tyrosine hydroxyl groups of other proteins using ATP as a substrate.
Proteolytic enzymes (proteases)
Enzymes that cleave proteins by a hydrolysis reaction.
Michaelis-menten enzymes
Enzymes that conform to simple Michaelis-Menten kinetics
Allosteric enzymes
Enzymes that regulate the flux of biochemicals through metabolic pathways
Phosphatases
Enzymes that remove phosphates added by kinases
Specificity
Enzymes won't recognize just any structure, only certain functional groups
Keq
Equilibrium Constant, indicates the tendency of a reaction to go toward completion, basically it is the rate forward divided by the rate reverse PRODUCTS OVER REACTANTS
Binding of Fatty Acids
Esterification
Where in eukaryotes one can find DNA?
Eukaryotes -Most located in the nucleolus -Some in the mitochondria and some in the chloroplasts as well -linear chromosomes that together make up the genome
Why Do We Need Enzymes?
Even if a reaction has a favorable ΔG and can occur spontaneously this rate may be too slow for a metabolic process (MAY TAKE TOO LONG FOR IT TO BE USEFUL TO THE CELL) so this is why enzymes are needed
Name the cancers caused by viruses.
Examples of Viruses Causing Cancer in Human: Hepatitis B and C (liver Cancer Human Papilloma Virus (Cervical Cancer HTLV-1 and HTLV-2 cause adult T cell leukemia and lymphoma -Sarcoma: cancer of connective tissue -Adenocarcinomas: cancers of glandular epithelial tissue
Formation of ES
First step of catalysis
Standard states
For gases, the gas is at a pressure of 1 atm. For solutions, a concentration of 1M, at 298K or 25C
Km
For most enzymes ______ lies between 10^-1 and 10^-7M, and depends on the particular substrate and on environmental conditions such as pH, temperature and ionic strength.
ΔG°
Free energy change under standard states (gasses-pressure at 1 atm) (solutions- concentration of 1 mol/L) (temp- degrees K) R=8.31J mol-1K-1
ΔG
Free energy for a reaction, reactants to products, defined in terms of enthalphy, entropy and temerature
Equilibrium Position
Function of the free energy difference between reactants and products (reactants and products themselves determine the direction not the enzyme)
Internal Energy (E)
Function that keeps track of the heat transfer and work expenditure in the system
Cholesterol Increase Fluidity
Fused ring structure is rigid and can disrupt regular interactions between the phospholipid tails and then increasing fluidity
G Protein Hormone Signalling
G proteins are lipid anchored, able to move laterally, this is important because it gives it the ability to move and attach to adenyl cyclase to create cAMP which is important in hormone signaling
techniques of inserting DNA to cells
GENE GUN -used to shoot DNA directly through thick cellulose walls -microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through plant cell walls MICROINJECTION -use glass micropipette with a diameter much smaller than cell that punctures plasma mem and inserts DNA through it
Irreversible Inhibitors
Generally involve covalent bond formation distinguished by a time-dependent decrease ine enzyme activity
Genetic Modification
Genes from one organism cell can be inserted and expressed in another organism cell -create useful products and application 1. Vector us isolated 2.DNA containig the gene of interest form a different species is cleaved by an enzyme into fragments 3.The deserrved gene is selected and inserted into a plasmid 4. The plasmid is taken up by a cell such as bacterium 5.Cell with the gene of intrest are cloned with either of 2 goals in mind( create and harvest copies of gene or create and harvest proteins product of gene)
E. Coli
Genetically Modified to Produce Gamma Interferon, a Human Protein That Promotes an Immune Response helps stop the attack,protein that fights virus
Brain
Has hexokinase, brain has a high demand for glucose this is why hexokinase is here because at very low [S] it will phosphorylate glucose, if the brain has a high Km enzyme it would be deprived of glucose because we have to wait for a very high concentration of substrate before phosphorylation occurs/enzyme is active
q
Heat absorbed by the system
Activators
Heterotropic effectors that shift the sigmoidal curve to the left
Inhibitors
Hetertropic effectors that shift the sigmoidal curve to the right.
What Two Enzymes Catalyze Glucose
Hexokinase and Glucokinase
Choleterol
High content in PM has steroid nucleus has important effects on PM plants/bacteria do not have this ONLY ANIMAL
The role of Histidine 57 in chymotrypsin activity
Holding Hydrogens for later donation to the N-terminus of one half of the hydrolyzed protein, and the reprotonation of serine 195 upon rearrangement in the second tetrahedral intermediate.
1 to 10^4 per second
How fast do most enzymes with their physiological substrates turnover?
Gibbs Free Energy (G)
How much energy is available to do work
Affinity labeling
How the importance of chymotrypsin's Histidine (the second residue involved in catalysis) was discovered.
Michaelis Menten...
Hyperbolic graph, substrate concentration on X and velocity of reaction on Y
Regulation
IMPORTANT, most reactions in cells do not happen at a constant rate, very important Many diseases are caused by failure to regulate enzymes properly (Cancer)
How Far the System is from Equilibrium
If there happens to be SOO many of some of the product the ΔG is going to be large because we do not need the product this is wronghahah
Why is Low Km Advantageous to Cell?
If you have limited substrate concentration you do not have to up this concentration for product to be made Osmotic effects, uses the glucose at small concentrations to kind of balance it if this didn't happen there would be too high blood glucose and fluid from the cells would leave by osmosis and go into the blood causing HTN
Transcription in Eukaryotes
In Nucleis and translation is in cytoplasm
acetylation
binding to N terminus
alkylating agents and acridine derivatives have in common...
both cause miss-pairing or frameshift mutations damaging DNA 1. alkylating agents add alkyl group and damage DNA 2. acridine derivatives insert into DNA ladder between backbones and damage DNA
DNA polymerase and RNA polymerase
both have DNA as template, look alike, both 5' to 3'...DNA polymerase makes DNA (involved in replication) where RNA polymerase makes RNA (involved in transcription)
opportunistic pathogen
an organism that exists harmlessly as part of the normal human body environment and does not become a health threat until the body's immune system fails
vector
an organism that transmits a disease or parasite from one animal or plant to another.
zoonosis
animal origin, disease spreads to human (ex. rabies, west nile virus, plague)
factors involved in decrease of mortality rate over last 100 years
antibiotics, vaccines, and cleaner water
viral growth measurement in animal cells
antiviral proteins=interferon, antibodies (proteins produced by immune system)
role of viruses in cancer
cancers caused by viruses: HepB, epstein-Barr, HTLVI, papilloma, herpes 20% cancers caused by viruses ONCOGENIC VIRUSES -cause host cells to transform and thus cause cancer -they control cell division, unless something causes damage to it, then its uncontrolled -they incorporate themselves into host cells' DNA and make uncontrollable cell growth -mammalian viruses -capable of starting tumors RETROVIRUS -special type of RNA virus that carries their own enzymes to make DNA from RNA -can cause cancer by taking oncogenes from a virus and incorporating it into a host -sometimes hosts bring own oncogenes, sometimes they affect the hosts' oncogene, sometimes that make proteins that encourage tumor growth
vehicle transmission
carriers disease inside you (food or water)
vector transmission
carries disease under your skin (flies, mosquitoes, insects in general)
reverse transcriptase
catalyzes formation of DNA from an RNA template (RNA to DNA); important for viruses because a virus with RNA must covert to DNA if host has DNA
transforming infection
causes host cell to become malignant
exonuclease activity
chopping/cutting DNA back, editing and proof-reading DNA
DNAs location in eukaryotes
chromosomes found in nucleus; extra chromosomal DNA found in mitochondria and chloroplasts
persistent infection
chronic infection that eventually clears
syndrome
collection of signs and symptoms
protoplast fusion
combining of 2 cell membranes together
genome
complete set of chromosomes and genes in an organism
Virion
complete, fully developed viral particle
Icosahedra
consisting of identical subunits that make up equilateral triangles that are in turn arranged in a symmetrical fashion.
Why Does not Everyone Benefit from Omeprazole?
cytochrome P450 metabolizes it and this base is prone to substitutions so when this is changed the body cannot metabolize omeprazole
CPE
cytopathic effect; structural changes in a host cell resulting from viral infection ex. lysis
Delta G/dG
dG^o - RTlnKeq
Less than 0, spontaneous, released
dG^o' for the hydrolysis of ATP. As a result, this process is ______ and energy is _______.
Zero, = (equal to)
dG^o' is ____ starting with 1M components and the reaction is at equilibrium. Keq _______ 1.
Positive, < (less than)
dG^o' is ____ starting with 1M components the reaction proceeds in reverse; Keq ______ 1.
Negative, > (greater than)
dG^o' is _____ starting with 1M components and the reaction proceeds foreward. Keq _____ 1.
Lipid Addition
determines where proteins end up, how a protein knows where it belongs, tag protein to a membrane by adding fatty acid chain
conjugation (HGT)
direct contact, DNA copied from donor and transferred via pili, vector=plasmid (ex. toxin genes, virulence genes)
reservoirs for rabies
dog, fox, skunk, jackal, wolf, cat, raccoon, mongoose, bat, rarely rodent or rabbit
replication
double helix is unwound with helicase, needs primer to start, is straightened and copied, DNA polymerase synthesizes DNA (semi-conservative, bidirectional, 5' to 3' direction antiparallel, circular chromosome, 2 complimentary strands); TEMPLATE= OLD STRAND; creates two DNA molecules
prokaryotic ribosomes
easier targets of antibiotics, smaller size
ligase
enzyme that fills gaps and fuses things together on strands
helicase
enzyme that opens strands and unzips DNA helix
characteristics of skin that make it resist infection
epidermis sheds so no permanent home, sweat glands make salt, cooler temperatures, lower pH (all are inhibitors)
five stages of disease
exposure, acquisition, progression, complication, death/disability
plasmid
extra chromosomal genetic unity, double-stranded DNA that is smaller and replicates independently, bears genes not essential for cell growth, can bear genes that code for adaptive traits, transmissible to other bacteria
RFLP
finger printing; forensic technique that takes human DNA, uses restriction enzymes and runs through gel to examine patterns and look at DNA from different sources (ex. paternity tests)
vertical gene transfer
genes transferred from parent to daughter cells (ex. humans)
pandemic
global disease outbreak, spread across large region
exotoxins
gram +/-, polypeptides, high toxicity, highly immunogenic, can convert toxoid (ex. botulism, tetanus) (polypeptides)
endotoxins
gram -, lipids, nonspecific ache-all-over, weak toxicity (lipopolysaccharides)
direct contact transmission
handshaking, kissing, sex
virulence
harmful quality possessed by microorganisms that can cause disease; virus has tools and structures to survive
chronic carrier
has been sick in past, now carries disease as host for a long period of time (doesn't have to show signs)
three examples of immunocompromised individuals
have HIV/AIDS, are pregnant, are going through chemotherapy, certain cancers, certain genetic disorders
medical, industrial, agricultural applications of recombinant DNA
herbicide resistance, salt tolerance, freeze resistance, pest resistance, improvements in nutritional value and yield, recombinant plants and animals
morbidity rate
how often a disease occurs in a specific area or is a term used to describe a focus on death (ex. number of people who have cancer)
reservoirs for influenza
humans (occasional bird, pig, ferret)
Viruses may attack which groups of organisms... Humans, animals.............
humans, animals, plants, bacteria
three approaches in epidemiological study
incidence, distribution, and possible controls of disease
transformation (HGT)
indirect contact, DNA picked up from environment, naked/free DNA, found in laboratories (ex. genetic engineering in labs)
transduction (HGT)
indirect contact, with the help of a virus it attacks the host and accepts DNA, virus=vector (ex. natural, happens often)
nosocomial infection
infection acquired at a healthcare facility; causative agents=staff, equipment, doctors, etc. (most common= UTI)
ID50
infectious dose
RNA interference (RNAi)
inserts DNA encoding siRNA into a plasmid and transferred into a cell
Zymogen activation
irreversible conversion of an inactive precursor to active enzyme by cleavage of covalent bonds
Transforming Viral Infections
is the change in growth, phenotype, or indefinite reproduction of cells caused by the introduction of inheritable material.
archaea
lack peptidoglycan, use different energy sources
eukaryotic ribosomes
larger size, more resistant to antibiotics
latent infection
last the lifetime of the host
notifiable diseases
law says must report to government (ex. anthrax, botulism, west nile virus, AIDS)
LD50
lethal dose
Envelope
lipid, protein, and carbohydrate coating on some viruses
symbiosis
living in close association 1. mutualism: benefits both 2. commensalism: benefits one/ doesn't bother the other 3. parasitism/antagonism: benefits one/ harms another
RNA molecules
mRNA= contains codon, codes for protein tRNA= contains anticodon, carries first amino acid rRNA=helps synthesize protein
Subunit vaccine
made from pathogen proteins in genetically modified yeasts
ribosomes
made of RNA and proteins
DNA Ligase
makes covalent bonds to join DNA strands and Okazaki fragments( short, newly synthesized DNA, lagging strand) and excision repair
carcinogens
molecule that may cause cancer
tissue culture/cell culture/monolayer
monlayer= layer of cells that grown on bottom, animal tissue is used if host is animal because primary cultures don't last long, virus needs host (so need host tissue)
MMWR
mortality and morbidity weekly report (CDC)
fungi
multicellular eukaryotes, cell wall made of chitin
components of virus
must have capsid (protein coat), and nucleic acid (DNA core), can have envelope and spikes and metric proteins
obligate intracellular pathogens
must have host to multiply, reproduce, replicate, etc. (viruses), other infectious agents include prions (pathogen that causes disease in animals)
induced mutation
mutations or mistakes in base pairings that go unfixed and are passed on daughter cell
virus shapes
naked helical capsids, enveloped helical capsids, naked icosahedral capsids, enveloped icosahedral capsids, complex capsids (most unique, ex. bacteriophage)
spontaneous mutation
natural base pair mistake by DNA polymerase and are fixed
reservoirs of infection
natural habitat for microbes (nature, water, lake, animals, humans; living and nonliving/animate and inanimate)
Virus Size
nm bacteria and Eukaryote are micrometer
Describe ONE-STEP growth cycle?
no virus detected(Eclipse period)> virus and host(virions realeased from host cell) acute infection *look at pic Binary Fisission(bacteria)
droplet transmission
not airborne, indirect
If enzymes are not maximally active, how is activity regulated
not all enzymes are active at once, cell can turn off enzymatic activity depending on what it senses in the environment enzymes can always be made but they are rapidly degraded and this changes, seems wasteful but it is a price to pay to make sure the cell is optimally regulated
viruses
not living, protein coat and nucleic acid core
DNA Polymerase
o Adds nucleotides to the growing DNA strand in the 5'-3' direction o Synthesizes DNA, proof reads DNA, repairs DNA o Removes mismatched bases before the synthesis process continues
What is the significance of reverse transcriptase?
o Artificial gene that contains only exons can be produced by using reverse transcriptase
Messenger RNA (mRNA)
o Carries the codes from the DNA to ribosome o Provides the information for making a specific protein occurs) o Carry Codon o Synthesized by RNA polymerase (recognizes the beginning of the gene, moves 5 to 3 prime) o mRNA is the translated version of the DNA (in the language of proteins) o contains uracil instead of thymine
Define/Explain/describe: Semiconservative replication
o Each new DNA molecule contains the original strand and one new strand o DNA is never completely brand new; always contains one parent strand and one daughter strand
Ribosomal RNA (rRNA)
o Forms an integral part of ribosomes o Machinery for protein synthesis o Ribosome is made up of rRNA and protein- it sandwiches the mRNA
What are ribosomes made of?
o Made of rRNA and protein o Every cell has them; used to synthesize proteins o Connect amino acids together to make a polypeptide chain o Sandwiches the mRNA during translation o Some are free floating in the cytoplasm and some are bound to the membrane of the endoplasmic reticulum
What is the source of reverse transcriptase (its origin)?
o Origin: Synthesizes complementary DNA from an mRNA strand template
Prokaryotes and eukaryotes have different ribosomes? What is the significance of this difference?
o Prokaryote: 70s ribosomes o Eukaryote: 80s ribosomes (60s subunit and 40s subunit) o Chloroplasts and mitochondria (in eukaryotes) have 70s ribosomes too ▪ Shows that prokaryotes evolved into eukaryotes over time
Define/Explain/describe: Complementary strands
o Strands that make up the double helix of DNA o They have complementary base pairs (A and T; C and G) o Complementary structure of the nitrogenous bases allows one strand to serve as a template during DNA replication
RNA Polymerase
o Synthesizes RNA from on strand of the double-stranded DNA (serves as template) o Binds to the promoter and allows transcription to begin o Recognizes the beginning of the gene and moves in the 5'-3' direction to synthesize an mRNA o Synthesis of RNA continues until the RNA polymerase reaches the terminator -recognizes the begining and end of gene
Define/Explain/describe: Anti-parallel
o The two strands of DNA are anti-parallel because they run in opposite directions o One strand goes from 5'-3' the other strand goes from 3'-5'
What does transcriptase do?
o mRNA then goes away through enzymatic digestion o DNA polymerase synthesizes a complementary strand of DNA- becomes double stranded o The cDNA that is produced through reverse transcription can be combined with the other molecules of cDNA from the same tissue or cell and be placed in the cDNA library o Popular method of obtaining eukaryotic genes o Only parts of desired gene may be transcribed if the mRNA
Define/Explain/describe: Double helix
o two strands made of nucleotides make up the double helix
fomite
object or substance capable of carrying infectious organisms (can transfer from one individual to another)
Define spontaneous mutation.
occur in the absence of a mutagen
endemic
occurs regularly
DNAs location in prokaryotes
one chromosome found in free area called nucleoide; extra chromosomal DNA found in plasmid
one-step growth cycle
penetration --> eclipse period --> release -eclipse period: period where the virus is not detected bc it injected their DNA inside the host. during this time biosynth and maturation happens. this is after penetration/attachment. before synthesis/assembly. -finally they start killing the hosts and the number of viruses grows on the growth curve
ribosome
peptide assembly site, coordinates codons of mRNA and tRNA anticodons, ribonucleoprotein structure
Mutation
permanent change in base sequence -can be neutral, harmful, and beneficial
Conjugation
plasmids transferred from one bacterium to another via direct cell-to-cell contact via sex pili 1. Replication and transfer of f factor to other bacteria 1. recombination btw f factor and chromosome occuring at specific site on each 2. insertion of the F factor into chromosome 1.Replication and transfer of part of the chromosome 2.in the recipient recombination btw the Hfr chromosome fragment and the F- chromosome f factor-fertility factor Hfr-high frequent recombnation
Cloning
population of genetically identical cells arising from one cell; each carries the vector
semiconservative replication
preserving the parent molecules by half old strand half new
semi-conservative replication
product is one old strand and one new strand
Spikes
projections from outer surface
Capsid
protein coat made of capsomeres (subunits)
translation
protein synthesis, happens at ribosomes; TEMPLATE= RNA; creates proteins
Introns
region of DNA that do not code for protein
Exon
region that codes for proteins
Topoisomerase
relaxes supercoiling ahead of the replication fork; separate DNA circles when DNA replication is done
Gyrase
relaxes the DNA strand before replication
release of endotoxin
released when bacteria dies, Lipid A causes inflammation, fever, drop in blood pressure, coagulation
Small nuclear ribonucleoprotein(snRNA)
removes intron and splice exon together
Gene Therapy
replace defective or missing genes
Name the tools used to make a recombinant DNA.
restriction enzyme,plasmid,host,original dna
Explain how a technique called RFLP works in analyzing DNA
restriction fragment length polymorphism • takes the genetic information of the nucleic acid/ DNA and cuts the DNA into pieces • southern blotting: DNA probes detect specific DNA in fragments (RFLPs) o DNA is first digested by a restriction enzyme -> creates thousands of fragments of different sizes o Fragments separated by gel electrophoresis o take DNA cut it up, put it in gel, transfer it to paper, a make a probe o probe binds to certain fragments and this can be seen by colored dye o genes can be tested for mutations o can be compared to the DNA from a father and child for a paternity test
ames test
screens for substances that may cause cancer
gene
segments of chromosomes that code for a specific protein or RNA molecule
Vector
self-replicating DNA molecule used to transport foreign DNA into a cell EX: Plasmid or virus
Shotgun sequencing
sequences small pieces of genomes which are assembled by a computer
horizontal gene transfer
sideways transfer of genes from one bacteria to another; conjugation, transformation, transduction (ex. bacteria)
Ribosome
site of protein synthesis in a cell made of RNA and protein
PCR
smaller extraction and amplification of DNA, every cycle DNA is doubled, good for crime scenes
carrier
someone who acts as a host for virus, carries it
sign
something I can see (ex. cut, bruise)
symptom
something I feel (ex. headache, pain)
codon
specific sequence of three nucleotides in mRNA that constitutes for a particular amino acid
DNA Polymerase
synthesizes DNA, proofreads and repairs
plague assay
technique used to isolate viruses, measures the ability of a single infectious virus to form on a plaque (plaque: clear spots on plates when one virus killed the host)
gel electrophoresis
technique uses electricity to form negative to positive current, and restriction enzymes to cut DNA into segments and the heavier travels slower, visual DNA
ΔG°' is Negative
the Keq is >1 think of it is because it is favored/normal so the reaction is going to proceed forward and make more product causing the numerator to be greater than the denominator
Allosteric constant (Lo)
the T/R ratio
frame shift mutation
the addition or removal of a base pair that changes the sequence and creates a nonsense molecule
etiology
the cause, set of causes, or manner of causation of a disease or condition
q
the heat absorbed BY the system
Recombinant DNA (rDNA)
the insertion or modification of genes to produce desired proteins
complementary strands
the nitrogenous bases pair up (A with T, C with G)
genetic code
the nucleotide triplets of DNA and RNA molecules that carry genetic information in living cells
mortality rate
the number of deaths due to a disease divided by the total population
anticodon
the trinucleotide sequence of tRNA that is complementary to the mRNA codon
Biotechnology
the use of microorganisms, cells, or cell components to make a product -Foods, antibiotics, vitamins, enzymes
anti-parallel
there is a 5' end and 3' end to DNA and run opposite of one another (5' to 3' direction)
chromosome
tightly coiled bodies in cells that are the primary sites of genes
neurotoxin
toxin that inhibits the nervous system (compound substances/elements)
enterotoxin
toxin that targets the intestines (protein)
transforming virus
transforming: infection transforms cells into malignant/tumor cells (i.e. cancer)
protoplast fusion
two species of plants are fused together to form hybrid plant with both characteristics, membrane fuses and contents mix, cell wall dissolves
lytic viral infection
type of viral infection that causes the cell to burst after invasion of virus
protozoa
unicellular, aerobic eukaryotes, cell wall made of cellulose
eubacteria
unicellular, peptidoglycan cell wall, larger of the two kingdoms
Concentration
universal mechanism, cell enzymes are regulated by [S], simplest way
Helicase
unwinds double stranded DNA , breaks the hydrogen bond
diseases outbreaks
usually caused by an infection, transmitted through person-to-person contact, animal-to-person contact, or from the environment or other media.
lysogenic infection
viral DNA becomes part of host cells DNA
viruses role in cancer
viruses interfere with genes that control cell division (cancers caused by viruses=oncogenes, cause uncontrollable growth; ex. retrovirus)
antigenic shift
when two or more strains of a virus combine, phenotype change, can do it with transposons or "jumping genes" from plasmid to chromosomes or chromosomes to plasmid
w
work done ON the system
Lineweaver Burk Plot
x is 1/[S] and y is 1/Vo
normal flora
yeast and bacteria
ΔE Equation
ΔE= q + w
Which Term is Relevant for Living Systems?
ΔG because we could never have a standard under standard, set conditions in the body because it is always changing and different
ΔG equation
ΔG= ΔH - TΔS
Enthalpy (H)
ΔH refers to the overall change in bond energy when bonds are broken or formed in a reaction, measure of the DISTANCE FROM EQUILIBRIUM OF A REACTION
2. Define Chromosome, gene, genome
• Chromosome: structures that contain DNA and physically carry the hereditary information , Contain genes • Gene: segments of DNA that encode functional products, usually proteins o in some viruses they are made out of RNA • genome: all of the genetic information in a cell. o Includes chromosomes and plasmids • Genome -> chromosomes -> genes
how to track the DNA
DNA fingerprinting is used to identify pathogens PCR microarrays and DNA chips can screen samples for multiple pathogens
recombinant DNA
DNA has multiple sources, a vector carries DNA into host (goals are to eliminate undesirable traits, combine good traits, and create organisms that synthesize beneficial products)
transcription
DNA is copied into RNA, uses DNA as template (initiation, elongation, termination); TEMPLATE= DNA; creates pre-mRNA
Transduction
DNA is transferred from a donor cell to a recipient via a bacteriophage -virus pass DNA from one bacteria to another Sometimes -Specific genes are transferred during this process: Specialized transduction (Specific bacterial genes are packaged inside a phage and transferred to a recipient cell) 1. a phage infects the donor bacterial cell 2.phage DNA and proteins are made and the bacterial chromosome is broken into pieces 3.occassionally during phage assembly pieces of bacterial DNA are packaged in a phage capsid. Then the donor cell lyses and release phage particle containg the bacterial DNA 4. A phage carrying bacterial DNA infects a new host cell, the recipient cell 5.Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells
genetic material for viruses
DNA or RNA, double or single stranded
fidelity of replication
RNA polymerase double checks strands for any errors, needs exact pair to match
1/s
Units of a first-order reaction rate constant
1/M*1/s
Units of a second-order reaction rate constant
Cis
Unsaturated fatty acid formation
Most fluid, decrease packing of the fatty acids
Unsaturated, cis
10^8 to 10^9 1/M*1/s
Upper limit of the Kcat/Km
Define frame shift mutation
-Insertion or deletion of one or more nucleotide pairs -Shifts the transnational "reading frame"
Translation
* Template: the mRNA o mRNA travels to the ribosome carrying the information to make a protein o mRNA carries the necessary codons o tRNA carries the anticodons and the amino acid to the ribosome o the sequences of the codons on the mRNA determines the sequence of the amino acids and the protein that will be created o each codon codes for an amino acid o there are 64 codons but only 20 possible amino acids (degeneracy) o start codon (AUG) codes for the beginning of protein synthesis o three nonsense codons (UAA, UAG, and UGA) signal the end of protein synthesis and do not code for an amino acid o the ribosome directs the binding of the tRNAs to the corresponding codons o an amino acid chain begins to be assembled and a protein is produced o as the amino acids are brought in line one at a time, peptide bonds begin to form making a polypeptide chain
Spontaneous Reaction
***You do not have to input energy to make a reaction happen!*** A reaction where the change in free energy is negative basically that the products have more free energy than the products so energy is released and it does not require an increase in energy to happen
virus vs bacteria
**look at chart VIRUS -DNA or RNA, ss or ds -100s of genes -nanometers -acellular -obligate intracellular parasite- require a host -invade all forms of life -infectious agent -capsid and nucleic acid, maybe envelope/spikes -no E source/replication -ubiquitous: found everywhere in nature BACTERIA -only have single, circular DNA -1,000s of genes -micrometers -MUCH LARGER THAN VIRUS CELLS
therapeutic applications of genetic engineering
**look at chart on slides -subunit vaccines: introducing antigen to cell w/out introducing viral particle; made from pathogen proteins in genetically modified yeast -human enzymes and other proteins like insulin -nonpathogenic viruses containing genes for pathogen's antigens like DNA vaccines -gene therapy to replace defective or missing genes -gene silencing: small interfering RNAs (siRNAs) bind to mRNA which is destroyed by RNA-induced silencing complex -RNA interference (RNAi): inserts DNA encoding siRNA into a plasmid and transferred into a cell
enzymes and their functions in DNA replication
**look at slide -helicase: unzips DNA by breaking H bonds -primase: synthesizes RNA primer, needed to start replication -DNA poly III: adds bases to the new DNA chain (5-3), proofreads chain for mistakes -DNA poly I: removes RNA primer, closes gap, repairs mismatch -ligase: finally fixes/binds DNA during synthesis and repair, binds okazoki fragments, IS NEEDED TO SEAL THE STICKY ENDS OF GENES INTO PLASMA OR CHROMATIDS
Transcription
*Template: the replicated DNA strand o RNA polymerase copies RNA from a DNA template o Genetic information from DNA is rewritten with the base sequences of mRNA o Process begins when RNA polymerase binds to the DNA at the promoter site o Only one of the DNA strands is used as a template o Synthesized in the 5'-3' direction o Synthesis stops when RNA polymerase reaches the terminator site on the DNA o Short term copies of the gene can be made which are directly used in protein synthesis o mRNA is created
Persistent Viral Infections
-A persistent viral infection occurs gradually over a long period; is generally fatal -Subacute sclerosing panencephalitis (measles virus) -Virus continuosly release
Families of Viruses That Affect Humans
-Human parvovirus B19>anemia in immunocompromised patients -Mastadenovirus>respiratory infections -Papillomavirus (human wart virus) Polyomavirus>warts and cervical and anal cance -Orthopoxvirus (vaccinia and smallpox viruses) Molluscipoxvirus>smallpox -Simplexvirus (HHV-1 and -2) Varicellovirus (HHV-3) Lymphocryptovirus (HHV-4) Cytomegalovirus (HHV-5) Roseolovirus (HHV-6 and HHV-7) Rhadinovirus (HHV-8)> chickenpox, shingles transcriptase to produce its DNA from mRNA; causes hepatitis B and liver tumors -Enterovirus Rhinovirus (common cold virus) Hepatitis A virus>polio -Hepatitis E virus Norovirus>causes of gastroenteritis -Flavivirus Pestivirus Hepatitis C virus>Can replicate in arthropods that transmit them -Filovirus> Ebola -Hepatitis D>Depend on coinfection with hepadnavirus -Influenza virus A, B, and C>Envelope spikes can agglutinate red blood cells - Reovirus Rotavirus>mild respiratory infections transmitted by arthropods
What is the role of viruses in cancer? Name the cancers caused by viruses.
-Several types of cancer are caused by viruses -May develop long after a viral infection -Cancers caused by viruses are not contagious -Oncogenes transform normal cells into cancerous cells -Oncogenic viruses become integrated into the host cell's DNA and induce tumors -A transformed cell harbors a tumor-specific transplant antigen (TSTA) on the surface and aT antigen in the nucleus
Describe how DNA can be inserted into a cells; By: Gene Gun,
-Shoot it directly in the thick cellulose -microscopic particle of tungsten or gold are coated with DNA and propelled bu a burst of helium through the plant cell wall. some cell will express the DNA as their own
Lysogenic Cylce
-Stages in viral development that result in the incorporation of viral DNA into host DNA. -ends when host dies -can cause diseases and antibioticc resistance
Uncompetitive inhibition
What sort of inhibition is the herbicide glyphosate demonstrating? (Roundup). Inhibits an enzyme in the biosynthetic pathway for aromatic amino acids in plants.
Virus
-Sub-microscopic, obligate intracellular parasites -With Host Specificity -Affecting Animals, Plants, Bacteria, Protozoa, Archaea Acellular Infectious Agents -Do not fit into 5 Kingdoms or 3 domains categories -Hallmarks of cell do not apply to viruses -Always need to be inside a host to multiply -Genetic materials can be DNA or RNA, ss or ds -Molecules on virus surface impart high specificity for attachment to the host cell -Upon infection virus affects host cell genetics and metabolism -Replication, transcription, and translation are directed to make virus particles. Viruses cannot synthesize ATP or their own proteins -Some carry genes to synthesize DNA or RNA using RNA as template (ONLY viruses carry such genes). Great tool in cloning
virus life cycle
-attachment: limitation of infection bc virus must be specific to host cell (some are highly specific, some not)- receptor is usually glycoprotein -penetration: inject only nucleic acid into the host -un-coating: sometimes the whole capsid gets inside along with the nucleic acid. in uncoating capsid comes off & nuc acid is released inside host -synthesis: synth of protein and nuc acids and replication -assembly: "maturation" or "packaging"; when virus pieces are put together to make a complete virus; eclipse period -release: either by killing the host or a portal of exit; usually 1 virus goes in and 100+ come out (envelope&spikes picked up on way out of host)
lytic cycle (virus type)
-bacteriophage infection that ends in lysis (killing of cell) -occurs in acute infection -when the cell dies, more viruses come out of it into the hosts body as a result -undergoes immediate replication/release -cycle results in destruction of infected cell in its membrane -virulent phage: phages that replicate only via the lytic cycle -unlike lysogenic, the viral DNA is separate w/ in the bacterial cell and replicates separately from the host DNA
What is the importance of Cyanobacteria
-blue green -unicellular -binary fission>filamentous forms( reproduce by fragmentation of the filaments) -like eukaryotic algae and are in the same environmental niche - Fix carbon dioxide, carry out oxygenic photosynthesis -can produce oxygen> photosynethetic cbacteria -Nitrogen fixation: nitrogen into ammonium (heterocysts) important to the development of like on earth when there was really little oxygen
Acyl-enzyme
What the first tetrahedral intermediate in the chymotrypsin catalytic process collapses to and remains as until it enters the second tetrahedral intermediate.
Binding Energy's Effect on Activation Energy
Binding energy (the energy released when the substrate binds to the enzyme) leads to the activation energy decreasing, because of the energy released the substrate is weaker and has a disruption of bonds so less energy is needed from the outside environment to break these substrate bonds so they can form new ones in the product
E + S <---> ES
Binding of the substrate
Glucokinase
Binds glucose because glucose has 2 OH groups one up and one down, WILL NOT bind galactose because galactose's OH groups are both oriented upward When galactose is present loses H bond capability
Negative Inhibitor
Binds to the R state and destabilizes it, thereby decreasing the concentration of R state allosteric enzyme.
Positive effector
Binds to the R state and stabilizes it, thereby increasing the concentration of R state allosteric enzyme.
Positive inhibitor
Binds to the T state and destabilizes it, thereby decreasing the concentration of T state allosteric enzyme.
Negative effector
Binds to the T state and stabilizes it, thereby increasing the concentration of T state allosteric enzyme.
In Animal Products
Body makes cholesterol accordingly, as needed in membrane, all steroid hormones are made from cholesterol also vitamin D
List agricultural applications of recombinant DNA.
Bt toxin Herbicide resistance Suppression of genes-Antisense DNA Nutrition-Human proteins Example: -Bt cotton and Bt corn: plants have toxin-producing gene from Bacillus thuringiensis; toxin kills insects that eat plants -Rhisobium meliloti bacterium: modified for enhance nitrogen fixation -Round up(glyophosphate0 resistant crops: palnts has bacterial gene, allows use of herbicide on weeds without damaging crops - Bovine growth hormone(bGH): improves weight gain and milk production in cattle; produces by E. coli -Cellulase: Enzyme that degrades cellulose to make animal feed stocks; produced by E. coli
The phases of the chymotrypsin reaction
Burst phase, steady-state phase
Sigmoidal
Curve demonstrated by an allosteric enzyme
Endonucleus
Cut DNA backbone in a strand of DNA; facilitate repair and insertions
Exonucleases
Cut DNA from an exposed end of DNA; facilitate repair
Compare Cyst vs. Trophozoite
Cyste -transmission inactive -very resistance to elements Trophozite -growing active and multiplying form -inside and grows
central dogma
DNA --> mRNA --> proteins --> function
how you introduce the DNA to the host cell,
DNA can be inserted into a cell by: -Transformation: Cells take up DNA from the surrounding environment -Electroporation: Electrical current forms pores in cell membranes -Protoplast fusion: Removing cell walls from two bacteria allows them to fuse gene gun, micrinjection, Ti Plasmid
Genotype vs phenotype
Genotype: the geneti make up of an organism Phenotype: expression of gene
What do we use to Predict the Direction of a Reaction
Gibbs free energy! (ΔG)
Liver
Glucokinase is here and is not very active until blood glucsoe levels rise, liver begins to take up some of the glucose to decrease the blood concentration of glucose that the rests of the body uses If we had a low Km enzyme in the liver blood glucose levels would be very elevated for too long because too many enzymes would all be making it
Induced Fit Example
Glucokinase, it has a cleft in the middle where glucose binds, significant conformational change occurs after the binding of glucose
Characteristics of Irreversible Inhibitors
Group specific, suicide inhibtors or not
Types of irreversible inhibitors
Group-specific reagents, affinity labels (substrate analogs), suicide inhibitors, transition-state analogs
vertical and horizontal gene transfer
HORIZONTAL -transfer of genes from one cell to another, doesn't come from other organisms -ex: arthropods, fomites, respiratory droplets, airborne disease -parenteral route: entry through injections, cuts, bruises- anything that directly deposits to body under the skin/mucous membrane VERTICAL -acquiring new genes from parent organisms to offspring -ex: via the ovum, sperm, placenta, milk -1 parent DNA to 2 daughter cells
Example of Gene Modification
Harvest copies of gene 1. gene encoding protein for pest resistance is inserted into plant cell 2.Gene encoding degradive enzyme to clean up toxic waste is inserted in bacterial cells Harvest proteins product of gene 1.Amylase, cellulase, and other enzyme prepare fabrics for clothing manufacture 2. Human growth hormone treats stunted growth
Integral proteins
Proteins that require strong detergents to remove. Are very difficult to investigate due to requiring a mostly non-polar environment.
Integral proteins
Proteins that span the membrane, and are strongly embedded in the membrane These don't need to go through the membrane entirely These are often seen with a high presence of alpha helices embedded in the membrane.
Stimulation of ATCase
Puring ATP shifts the curve left, positive modifier that increases the rate of synthesis, produces more at lower amounts of aspartate
Example; how you cut DNA,
REtriction enzyme cuts the DNA
Transfer RNA (tRNA)
Transport the required amino acids to the ribosome o Recognize the specific codons o Carry anticodons which are commentary to the codons on the mRNA
Zymogen forms of trypsin and chympotrypsin
Tripsinogen and chymotrypsinogen
Virus Induced Changes in Cells
Types of Virus-Cell Interactions -Permissive or productive infection -Abortive or non-productive -Persistent -Latent -Traansforming Cytopathic effects (CPE)
Characteristics of Transition State
UBSTABLE Results in maximal binding energy Results in release of this energy
Uncompetitive Binding
Uncompetitive inhibitor binds to the ES complex and forms ESI which then depletes ES
Multiplication and Inheritance Processes of the Retroviridae
Uses revese transcriptase 1. REtrovirus enter by fusion btw attachment and the host cell receptors 2. Uncoationg release the 2 viral RNA and the viral enzyme reverse transcriptase, integrase, and protease 3. Reverse transcriptase copies viral RNA to produce double stranded DNA 4. The new viral DNA is tansported in the host cells nucleaus where its integrated into a host cell chromosome as a provirus may be replicated whe the host cell replicates 5. Transcription of the provirus may also occur, producinf RNA for new retrovirus, capsid, enzymes, and envelope proteins 6.Viral proteins are processed by vira protease, some of the viral proteins are moved to the host plasma membrane 7. Mature retovirus leaves the host cell acquiring an envelope and attachment spike as it buds out Attachment * uncoating Retrovirus* PRovirus* synthesis* (protease inhibitor cant make more protein) assembly Exit * can be stopped or controled
virus vs prokaryotes vs eukaryotes
V-few hundred of genes P-thousands of genese E- tons of gened
List forms of fungi
Vegetative Structure -Yeasts Nonfilamentous and unicellular Budding yeasts divide unevenly Fission yeasts divide evenly -Dimorphic fungi Yeastlike at 37 C and moldlike at 25 C(different form at different temperature)
State the difference between and give examples of vertical and horizontal gene transfer?
Vertical gene transfer: transfer of genes from an organism to its offspring Horizontal gene transfer: transfer of genes between cells of the same generation Transformation Transduction Conjugation
Name different components of a virus. Name the two that every virus must have
Virion—complete, fully developed viral particle -Nucleic acid—DNA or RNA can be single- or double-stranded; linear or circular -Capsid—protein coat made of capsomeres (subunits) -Envelope—lipid, protein, and carbohydrate coating on some viruses -Spikes—projections from outer surface
K2
Vmax/[E]t
Kcat (turnover number)
Vmax/[Et]
Why is LB Plot Useful?
We can run enzyme assays to test drugs for drug design/development, enzyme kinetics useful for predicting effects of inhibitors on enzyme activity Enzyme assays, can see how a molecule is inhibiting an enzyme
Why Do We Care about Gibbs Free Energy (G)?
We care because in order to make bonds we need energy (gibbs free energy tells us how much energy we need) and how much energy is going to be given off when a bond is broken ALSO you can link reactions together depending on their delta Gs to make them happen
How Do We Fix This Equilibrium Problem?
We increase concentrations on different sides of equations through the use of coupled reactions
High Km
Weak binding between E and S, needs increased [S] to function, if you have too little substrate the reaction and the production of products will take forever
High Km
Weak binding, [S] around 10^-6M, need to increase [S]
Fluorescence Recovery After Photobleaching (FRAP)
What proved that membranes were fluid?
Define Central Dogma of Molecular Biology (list its chain of events)
• DNA -> mRNA -> protein formation -> functional proteins • Theory created by Francis Crick in 1956 1. A sequence of nucleotide in DNA determines the sequence of amino acids in a protein 2. Transcription: genetic information in DNA is copied, or transcribed, into a complementary base sequence of RNA 3. Translation: cell uses information encoded in the RNA to synthesize specific proteins • Describes how DNA becomes messenger RNA, which turns into protein 4. Becomes a functional protein • If a mutation occurs this process is altered o Mutated DNA -> mutated mRNA -> altered protein structure -> altered protein function (The change in the DNA sequence of amino acids alters the folding of the protein which alters its ability to function and the RNA)
Define catabolic Repression
• Inhibits gene expression • Decreases the synthesis of enzymes • a process that can override inducible operons • when you have lactose and glucose together the glucose is used first -> the inducible operon is turned off • the lag time between using glucose and lactose -> the time it takes to turn on operon • catabolic repression does not affect the repressible operons • inhibition of the metabolism of alternative carbon sources by glucose
What is meant by recombinant DNA (technology)?
• Recombinant DNA technology: the insertion/ modification of genes to produce desired proteins • DNA from two different sources are put into one cell • also known as genetic engineering
Explain Operon's role in gene expression (transcription) in bacteria.
• Set of operator and promoter sites and the structural genes that they control • Regulated by the production of regulatory genes • Operator: provides the go and stop signal for transcription of structural genes • Promoter: place where RNA polymerase begins transcription • Regions of DNA that code for multiple genes • Genes are expressed only as needed
Define Operons
• The process of turning genes on and off can save energy for the cell- only making what is necessary at the time
What is the genetic material for viruses, DNA or RNA, single stranded or double stranded?
• Viruses can have DNA or RNA, but can't have both • Can be double stranded or single stranded • Linear or circular
Describe the use of Recombinant DNA in Producing Insulin.
• bacteria with genes for human insulin can be used to produce insulin for diabetic patients • vector is introduced to the bacteria and begins to multiply creating a clone • vector: self-replicating DNA molecules used to transport foreign DNA into a cell Ex: production of insulin o the insulin they used to get from animals was not as effective as the insulin they can now produce from humans o synthetic genes were constructed for the two polypeptide chains that are present in insulin o synthetic genes introduced to a plasmid (vector) o these synthetic genes were linked to genes that were already coding for a different gene (beta-galactosidase enzyme) o allowed the insulin enzyme to be produced at the same time as the other enzyme o an E. coli bacteria was used to produce each of the polypeptide chains o polypeptides were collected from bacteria and the beta-galactosidase enzyme was removed o two polypeptides were joined by chemicals to make the human insulin
What is the purpose of cloning?
• clone: population of genetically identical cells arising from one cell; each carries the vector • from this clone researchers can harvest large amounts of the gene they are interested in studying
