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Microbiology > Exam 2 > Flashcards

Exam 2 Flashcards

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1
Q

viral life cycle

A
  • attach
  • enter/uncoat
  • replicate/assemble
  • exit
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2
Q

infected call recognition

A
  • healthy cells express SELF proteins
    • not recognized by immune cells
  • viral protein on infected cells
    • killer T cell recognizes viral protein
    • perforin released by killer T cell and makes holes in the infected cell
    • granzyme enters cell through holes and causes apoptosis
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3
Q

Gene expression

A
  • DNA polymerase replicates DNA
  • RNA polymerase transcribes DNA into mRNA
  • Ribosome translates mRNA into a protein
  • protein provides a function
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4
Q

Genetic mutation as a cause of drug resistance

A
  • billions of bacteria will have few with mutations (spontaneous)
  • in the presence of the drug only the bacteria with advantageous mutations will survive
  • vertical transmission when surviving bacteria replicate
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5
Q

Selective Pressures

A
  • drugs
  • disease
  • prey/predator
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6
Q

Horizontal vs Vertical Gene transfer

A
  • horizontal is cell to cell
    • conjugation
    • transformation
  • vertical - mother to child
  • horizontal and vertical can spread antibiotic resistance
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7
Q

Nitrogenous Bases

A
  • cytosine and thymine are pyrimidines (monocyclic)
  • adenine and guanine are purines (dicyclic)
  • CG pairs have 3 H bonds
  • AT and AU pairs have 2 H bonds
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8
Q

DNA structure

A
  • nucleotide - sugar, phosphate, nitrogenous base
  • phosphodiester bonds between nucleotides
  • sugar phosphate backbone
  • has directionality
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9
Q

DNA vs RNA

A
  • double and single stranded
  • 2’ deoxyribose for DNA and ribose for RNA (2’ hydroxyl)
  • both have directionality
  • ATCG (dna) and AUCG (rna)
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10
Q

Operon

A
  • series of adjacent genes regulated by the same promoter
  • produces one long mRNA that is polycistronic
  • genetic content of many genes
  • promoter comes before the operon
  • often genes in an operon are for a common pathway
  • often all genes needed for a protein are in the same operon (ex Capsule - enzymes and transporters)
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11
Q

promoter

A
  • sequence of DNA that recruites RNA polymerase
  • may initiate synthesis of a monocistronic or polycistronic mRNA
  • The promoter is not part of the mRNA that is used to make the protein
  • RNA polymerase begins at the transcription start site that comes after the promoter
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12
Q

open reading frame

A
  • genetic sequence between the start and stop codons
  • often has all of the genetic info needed to make a protein
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13
Q

Prokaryote vs eukaryote genes

A
  • prokaryotes
    • no nucleus (DNA in cytoplasm)
    • no introns
    • poly A tail destabilizes
    • no caps
    • coupled processes
  • Eukaryotes
    • DNA in the nucleus
    • introns spliced out
    • no cap
    • poly A tails
    • 5’ caps
    • uncoupled processes
    • RNA to cytoplasm
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14
Q

Regulator Protein

A
  • aka transcription factor
  • can bind to promoters and operons on different parts of a chromosome
  • proteins are related
  • regulon - group of operons that are controlled by the same regulator (can turn genes on AND off)
  • promoters/operons can be on different positions on the chromosome
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15
Q

3 regulators of DNA replication initiation

A
  • DnaA-ATP conc high
    • 4 molecules bind to 9mer
  • low concentration of bound SeqA (inhibits initiation)
    • binds to hemimethylated DNA
  • methylation status of oriC
    • oriC must be fully methylated (adenines)
    • hemimethylated DNA cannot be replicated
    • Dam methylase methylated hemimethylated DNA
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16
Q

Replication initiation components (1)

A
  • oriC - origin of replication
    • 9mer and 13mer
  • 4 molecules of DnaA-ATP bind to 9mer. ATP bends the DNA and 13mer unwinds. Now single stranded
  • helicase loader (DnaC) loads helicase (DnaB) onto 13mer (ssDNA)
    • 2 helicases bc bidirectional
  • Helicase unwinds DNA. Recruit primase
  • Primase synthesize an RNA primer for continuous leading strand
  • DNA poly III uses 3’ OH to start synthesis
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17
Q

Replication initiation components (2)

A
  • clamp loader attachs sliding clamp at the RNA primer
  • DNA poly III attaches to the sliding clamp
    • this happens several times on the lagging strand
  • replisome - (2) DNA poly III, sliding clamp, clamp loader
  • 2 replisomes per nucleoid
  • 2 DNA poly III - each synthesize a single strand
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18
Q

DNA polymerase III

A
  • synthesiizes DNA during replication
  • fidelity - how faithfully DNApolyIII uses temlpate stand, how error prone
  • processivity - how fast replication occurs, how long it stays on the template.
    • determined by the sliding clamp
  • proofreading - fixes mistakes
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19
Q

Elongation of Replication

A
  • several RNA primers on lagging strand
  • Single stranded binding proteins (SSB) - keeps ssDNA elongated and stabilizes it, no degradation
  • lagging strand - every 1000 basepairs new RNA primer, new DNA poly III and clamp
    • continue till hit previous okazaki fragment then release DNA poly III and clamp
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20
Q

Remove RNA primer

A
  • RNase H or DNA poly I cleaves RNA section
  • DNA poly I replaces RNA with DNA
  • DNA ligase repairs phosphodiester backbone
  • occurs more on the lagging strand, but occurs on both
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21
Q

Termination of Replication

A
  • ter sequences are in both directions
  • multiple terminator sequences
  • Tus proteins bind to ter sequence to stop helicase
  • Topoisomerase IV breaks linked circles and puts them back together
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22
Q

Initiation of Transcription

A
  • promoter is the specific DNA sequence where the RNA poly should bind
    • sigma factor (transcription factor) determines what promoter RNA poly binds to. Holoenzyme scans the DNA
    • promoter at -35 and -10 site. Not part of the transcript
  • Closed complex when RNApol binds and sigma factor is still associated. No transcription
  • drop sigma factor and change conformation of RNA poly. Single strand DNA
  • Open complex - DNA unwinds and transcription begins at the transcriptional start site (+1)
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23
Q

Core and holoenzyme

A
  • RNA polymerase
  • core enzyme - (2) alpha, beta, beta’
  • holoenzyme - (2) alpha, beta, beta’, sigma factor
  • sigma factor - guide RNApol to different promoters
    • corrdinated control of the genes in similar process (all proteins required for sporulation)
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24
Q

How do several promoters with different sigma factors code for the same genes?

A
  • multiple promoters upstream of the operon
  • varying lengths of mRNA produced
  • translation will always start at the same start codon
  • example: heat stress, osmotic stress, pH stress, starvation all initiate chaperone proteins
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25
Q

Consensus Sequence

A
  • when promoters look similar and are often related/similar function
  • nucleotide/amino acid that is most frequently found at the position in the sequence
  • will be found in promoters that have related functions
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26
Q

Termination of Transcription

A
  • Rho dependent termination
    • stall RNApol due to GC rich area
    • Rho binds GC rich sequence in mRNA
    • mRNA wraps around Rho and when Rho contacts RNApol termination occurs
  • Rho independent termination
    • NusA binds GC terminator stemloop
    • RNApol hits NusA and hairpin and termination occurs
    • Uracil rich after GC stemloop because only 2 H bonds and RNApol drops easier
    • GC stemloop, NusA binds, consecutive uracils
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27
Q

CpsE

A
  • first enzyme that starts the process of capsule synthesis
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28
Q

Codons

A
  • one start codon AUG
  • Wobble base - third position in codon. several codons can code for the same amino acid
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29
Q

tRNA

A
  • interpret the codon
  • has an anticodon that is complementary to the codon in the RNA
  • tRNA is “charged” when it is attached to an amino acid
  • “uncharged” when not attached to an amino acid
  • amino acid binds to the acceptor end of the tRNA
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30
Q

Ribosome

A
  • made of RNA and proteins
  • ribozyme - RNA enzyme
  • 23S most important subunit - part of 50S, peptidyltransferase (forms peptide bonds between AAs)
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31
Q

Initiation of translation (RBS)

A
  • ribosome recognizes specific sequence (RBS-ribosome binding site)
    • Shine-Dalgarno
  • consensus sequences are conserved in related strands
  • binds to mRNA
  • start codon marks the start of translation and dictates the reading frame
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32
Q

5’ Untranslated Region

A
  • mRNA region between the transcription start site and the start codon
  • variable lengths can allow for several promoters to code for the same protein
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33
Q

Initiation of Translation

A
  • IF3 guides 30S subunit to mRNA and blacks premature 50S docking
  • IF1 blocks A site to prevent premature loading of tRNA
  • IF2 guides fMet-tRNA to P site and IF3 leaves
  • IF1 anf IF2 leave as 50S associates
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34
Q

Ribosome sites

A
  • A- acceptor site - entry of hte charged tRNA
    • complementarity of the codon and anticodon determines if the tRNA enters the site
  • P - peptidyl-tRNA - AA is transferred to the growing polypeptide
  • E - exit - tRNA without AA leaves the ribosome
35
Q

Translation Elongation

A
  • charged tRNA enters the A site
  • AA linked to chain at the P site
  • uncharged tRNA exits at E site
  • Occurs in a translocation movement
    • energy dependent of ribosome along the mRNA. 5’ to 3’
  • N terminus is at the 5’ end of the mRNA
36
Q

Translational inhibiting antibiotics

A
  • bacteriostatic drugs
  • tetracycline blocks A
  • streptomycin blocks fMet-tRNA
  • chloramphenicol and erythromycin bind to 50S
37
Q

Translational Termination

A
  • Stop codons are used
  • No tRNA with anticodon that matches the stop codons
  • release factor enters the A site and the ribosome disassembles
38
Q

Mutations

A
  • Point mutations: single nucleotide change
    • silent - no change in AA (wobble base)
    • missense - change in AA
    • nonsense - early stop codon
  • frameshift - insertion or deletion will cause every codon to change
39
Q

Single mutation impact on pathogenicity

A
  • Transparent cells lacked a capsule and were not pathogenic
  • opaque cells had a capsule and were pathogenic
  • Transparent cells were not recovered and the mice survived
40
Q

protein secretion (5 types)

A
  • SRP mediated transertion - insert into cell membrane
  • General sec pathway - secretion unfolded proteins
  • Twin arginine translocase (TAT) - pre folded proteins
  • Type I secretion system (T1SS) - ABC transporter across IM and OM (mosty Gram -)
  • T3SS - pathogens, inject protein into a host. Evade immune detection (mosty Gram -)
41
Q

SRP mediated transertion steps

A
  • signal recognition particle
    • Ffh protein and ffs sRNA
  • uses hydrophobic sequence as a Nterminus signal sequence
  • SRP binds to hydrophobic region and ribosome stops
  • SRP delivers paused ribosome to FtsY (membrane protein)
  • Protein threaded through SecYEG (transmembrane pore) translocon into the membrane or direct insertion into IM
    • both pathways require FtsY
42
Q

SRP general info

A
  • insert protein into membrane before translation is complete
  • coupled translation and insertion
  • “nascent” - newly synthesized protein
  • Direct insertion or SecYEG pathway determination is unknown
43
Q

Sec mediated secretion

A
  • ribosome completely translated the protein befoer exported
  • SecB winds around protein
  • Delivered to secYEG translocon
  • ATP used by SecA (SecYEG subunit) to power export of protein through SecYEG
    • about 1 ATP per 20 AA
  • LepB cleaves a signal sequence and releases protein from SecYEG.
  • Cleavage of sig. sequence allows protein fold outside of cell
44
Q

Twin Arginine Translocase

A
  • secrete folded proteins
  • RRXFXK Motif (signal sequence) is the protein recognized for transport by TAT
  • Uses TatA TatB and TatC
  • RR bound by TatC and recruite TatB and TatA
  • TatA molecules make the pore
  • No ATP. Powered by the proton motive force
45
Q

Type III Secretion System

A
  • Almost exclusively Gram (-)
  • Pathogens inject protein from bacteria into cytoplasm of host
  • Example - Yersinia pestis - bubonic plaque
    • injects into phagocytes
    • blocks immune cell activation pathways
    • paralyze cell and prevent it from expanding in order to phagocyte
  • large swollen lymph nodes due to excessive bacteria blocking lymphatic vessels
46
Q

Type I Secretion System

A
  • Export to extracellular space
  • tunnel from cytoplasm to extracellular space
  • HlyB in the IM and has ABC which hydrolyses ATP
  • Tunnel is made of HlyD
  • TolC spans the OM
  • Almost exclusively Gram (-)
47
Q

Transformation Brief Intro

A
  • Uses a transformasome
  • One strand of dsDNA is degraded and ssDNA enters the cell (DNA degrading enzyme)
  • Free DNA is incorparated into the chromosome
48
Q

Detailed Transformation Steps

A
  • ComC-CF precursor produced. Active CF exported via ComA and ComB. Requires ATP
  • When cell neighbors increase, CF increases (quorum sensing)
  • [CF] must be high enough to bind to ComD and be brought into the cell
  • ComD phosphorylates (from ATP) ComE and begins the phosphorelay transfer of P
  • Activate ComX gene which codes for SigH
  • SigH is sigma factor for transformasome genes
  • Transformasome brings DNA into the cell
49
Q

About Transformation

A
  • Transformasome makes the cell competent and able to uptake naked DNA
  • DNA must be incorporated into the genome after it is in the cell
  • CF is a positive feedback loop
  • naked DNA - noncomplexed
50
Q

Noncompetent cells

A
  • Can become competent without a transformasome
  • uses electroporation, chemical transformation, pili
51
Q

Quorum sensing

A
  • chemical communication
  • cell knows how many neighbors based on [CF]
  • coordinates gene expression
  • saves energy by only making transformasome when neighbors and around and naked DNA is likely.
52
Q

Type IV Secretion System pili Transformation

A
  • Binding of dsDNA to pilus
  • disassembly of pilus to bring dsDNA to ComE (DNA binding protein)
  • Then to ComA (inner membrane pore) then the cytoplasm
  • Becomes single stranded
  • recombine into the genome
53
Q

Bacterial Conjugation Steps

A
  • donor sex pilus attaches to recipient receptors
  • contraction draws them together. Relaxosome bridge
  • F factor nicked at oriT. 5’ end transfers
  • Strand in the donor is replicated
  • Transfer strand circularizes and replicates
  • Bridge disassembles and seperate.
  • Now both are F+
54
Q

Conjugation Info

A
  • relaxosome - connects the cells
  • tra genes - encode proteins that form the relaxasome
  • sex pilus - makes initial contact to recipient
  • episome - plasmid DNA can exist alone or integrated into the chromosome
  • endonuclease - nicks oriT
  • F plasmid genes - contains accessary genes and genes to make F pilus and relaxosome (tra genes) and endonuclease
  • oriT - begin transferred strand
  • oriV - nontransferred plasmid origin
  • rolling circle (unidirectional) replication
55
Q

General Transduction Steps

A
  • phage infects bacterium
  • DNase cut up host DNA, reduce competition
  • Synthesize phage parts
  • DNA into capsid. Some parts will be bacterial DNA
  • Phage assembly
  • cell lyse and phage released
  • Transducing phage particles inject host DNA into a new cell
  • Recombination crossover events exchange host DNA for donor DNA
56
Q

General Transduction Info

A
  • transfer any gene, do not have to be connected to phage DNA
  • transducing particle - phages that carry host DNA
    • dead end for phage life cycle - carries bacterial DNA, is not replicating its own genetics, no new phages produced
  • lysogeny - phage replication in which gene is incorporated into the host
  • capsid contains the DNA
  • DNase - cut up host DNA
    • reduces detection
    • increases virulence
    • reduce competition for replication and growth machinery
    • increase chance that host DNA is packaged into capsid
57
Q

prophage

A

-phage DNA that is incorporated into the bacterial genome

58
Q

Specialized Transduction Steps

A
  • temperate phage injects DNA
  • Prophage DNA - phage DNA into bacterial chromosome
  • When prophage DNA is removed, may take bacterial DNA with it
  • prophage DNA w/bacterial DNA is packaged into the capsid
  • lysis - release phages
  • Injected into new host and bacterial DNA is recombined into host along with prophage DNA
59
Q

Specialized Transduction Info

A
  • temperate phage - under goes lysogenic replication
  • genes must be close together/attached
  • prophage DNA is connected to bacterial DNA
  • fewer genes transferred
  • lysogenic replication
  • Not a dead end for transducing particles
60
Q

General vs Specialized transduction

A
  • both - new genotypes, phage is used to transfer to new bacteria, transducing particles
  • generalized - all bacteria DNA susceptible to transfer, genes do not need to be connected to phage DNA
  • specialized - genes must be close/attracted
61
Q

Restriction modification steps

A
  • DAM methylase methylates host DNA near recognition sites in order to keep restriction endonuclease from thinking its foreign
  • restriction endonuclease uses recognition sites to identify foreign DNA then cleaves it
62
Q

Restriction Modification Info

A
  • endonuclease - cleave nucleic acids in the DNA
  • exonuclease - cleave nucleic acids at ends of DNA
  • DAM methylase - add methyl to adenine
  • recognition sites - palendromic repeats that alert restriction endonuclease
63
Q

How to stop the lytic cycle

A
  • degrade phage DNA before replication - restriction modification
  • modify host range and avoid tail fibers
    • mutate LPS to avoid receptors
  • Mutate receptor
  • CRISPR
64
Q

CRISPERCas Steps

A
  • Phage injects DNA - immunization
  • part of phage genome inserted as a spacer - immunization
  • guide RNA (crRNA) transcribed from spacer. Repeat also transcribed and forms stemloops
    • stemloops stabilize crRNA - expression
  • Form complex with cas nucleases - looks for familiar DNA that has been seen before - interference
65
Q

CRISPRCas info

A
  • sequence specific immune system against phage
  • repeat - palindromic - form stem loops
  • spacer - genetic info from phages
  • cas genes - encode nucleases that degrade nucleic acid
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • crRNA forms complex with cas protein
66
Q

CRISPR Application

A
  • allows for site specific DNA editing
  • disease
  • increase ag production
  • animal modification
  • increase resistance to bacterial infection
67
Q

Recombination Advantages

A
  • acquire advantageous genes (metabolic diversity, antibiotic resistance, toxins, fimbriae)
  • increase genetic diversity
  • fix mutations
    • either from endogenous DNA from replicated chromosome or exogenous DNA from donor
      *
68
Q

Newly acquired DNA fate

A
  • plasmid DNA - independent
  • linear DNA - must be recombined
  • fails and is degraded by nucleases
  • horizontal gene transfer
    • plasmid - conjugation
    • linear - transformation, gen/spec transduction
69
Q

Generalized Recombination of 2 circular DNA

A
  • F plasmid is independent
  • sometimes plasmid can recombine into genome
    • requires sequence homology (stretch of DNA on plasmid that is virtually identical to chromosome)
    • insertion sequences
  • single crossover event
    • one large piece of DNA = plasmid + chromosome
  • the recombinant/co-integrate is the DNA of the combined plasmid and chromosome
70
Q

2 circular recombination steps

A
  • RecBCD proteins bind to dsDNA and unwind due to helicase component. Nicked to create ssDNA region (endonuclease activity in RecBCD)
  • RecA coats ssDNA
  • RecA filament scans chromosome for sequence homology between ssDNA and chromosomal dsDNA
    • aligns DNA
  • RecA allows filament to displace a strand of DNA
  • DNA triplex (synapse) forms
  • recombination occurs
71
Q

Generalized Recombination (linear and circular)

A
  • ssDNA in cytoplasm, bound to DprA
  • DprA recruits RecA
  • Strand invasion and form synapse
  • strand exchange
  • double crossover event
  • NO increase in DNA length, swap of genetic info (equal exchange)
  • higher sequence homology increases recombination frequency
    • at least 2 regions of sequence homology are required
72
Q

Mutation effects

A
  • neutral - no change in phenotype
  • gain - advantageous
  • loss - deleterious
  • knockout mutation - null mutation (no protein made)
73
Q

Point Mutations

A

transition - purine to purine or pyrimidine to pyrimidine

transversion - purine to pyrimidine or pyrimidine to purine

purine (A, G)

pyrimidine (C, T)

74
Q

Inversion mutation

A
  • double strand break and reverse order
  • wild type - most common, unmutated, original
75
Q

Reversion Mutation

A
  • change to mutation and then reverse to wild type
76
Q

Nucleotide excision repair (NER) steps

A
  • UvrA and UvrB form a complex and then bind to a thymine dimer
  • UvrA bends the DNA and is ejected
  • UvrB recruites UvrC
  • UvrC cleaves the phosphodiester backbone. Cuts out area larger than dimer
  • UvrD w/helicase unwinds and strips damaged DNA. leaving ssDNA
  • DNApol I fills gap. DNA ligase connects backbone
77
Q

Base Excision Repair

A
  • Uracil glycosylase removes damaged base. backbone intact
  • Apyrimidinic or apurinic (AP) site formed
  • AP endonuclease cleaves phosphodiester bond and frees up 3’ OH
  • DNApol I synthesizes replacement strand, while exonuclease activity simultaneously degrades
  • DNA ligase seal strand
78
Q

Quorum Sensing Steps

A
  • Luxi produces autoinducer
  • AI diffuses into medium and accumulates
  • At threshold, [AI] enters cell and binds to LuxR in the cell
    • iux operon initiated and transcription begins
  • Genes controlled by single operon
79
Q

Quorum Sensing Info

A
  • sense chemical environment to coordinate gene expression and synchronize group behavior
  • coordinate gene expression and produce toxins simultaneously in order to increase pathogenicity
    • ex. [CF] in transformation
  • Always produce AI at low level. Positive feed backloop when near neighbors that also secrete AI
80
Q

Vibrio Cholerae

A
  • Gram -, vibrio
  • flagellated
  • facultative pathogen - can cause disease but doesnt have to
  • can live in fresh water and human gut
  • sense environment in order to regulate gene expression
81
Q

Two Component System

A
  • In Gram + and - bacteria
  • Sensor kinase - binds to environmental signal and starts a phosphorelay
  • response regulator - when phosphorelated can bind to DNA and alter genes
  • cognate - sensor kinase and response regulator are a set pair
  • Vibrio example - in pond the sensor kinase binds to NaCl and response regulator leads to NaCl tolerant genes
    • in human - detect carbs and turn on carb uptake genes
82
Q

LacZYA operon components

A
  • lac Z - B galactosidase - enzyme cleaves lactose
  • lac Y - permease - transmembrane pore that imports lactose
  • lac A - transacetylase - unknown function
83
Q

About Lac Operon

A
  • glucose preferred carb source
  • inducible expression - can be turned on and off
  • repressor binds to operator between promoter and genes - stops RNApol from transcribing the genes
    • allolactose binds to repressor and releases it. Genes transcribed
  • CAP enhances transcription - CAP on when cAMP is bound (cAMP is low when glucose is high)
    • when CAP is inactive, RNApol is less effective
84
Q

Lactose and glucose levels chart

A

Microbiology (6 decks)

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