Exam 2 Flashcards
1
Q
viral life cycle
A
- attach
- enter/uncoat
- replicate/assemble
- exit
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
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
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
5
Q
Selective Pressures
A
- drugs
- disease
- prey/predator
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
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
8
Q
DNA structure
A
- nucleotide - sugar, phosphate, nitrogenous base
- phosphodiester bonds between nucleotides
- sugar phosphate backbone
- has directionality
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)
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)
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
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
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
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
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
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
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
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
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
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
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
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)
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)
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
25
Consensus Sequence
* 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
26
Termination of Transcription
* 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
27
CpsE
* first enzyme that starts the process of capsule synthesis
28
Codons
* one start codon AUG
* Wobble base - third position in codon. several codons can code for the same amino acid
29
tRNA
* 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
30
Ribosome
* made of RNA and proteins
* ribozyme - RNA enzyme
* 23S most important subunit - part of 50S, peptidyltransferase (forms peptide bonds between AAs)
31
Initiation of translation (RBS)
* 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
32
5' Untranslated Region
* mRNA region between the transcription start site and the start codon
* variable lengths can allow for several promoters to code for the same protein
33
Initiation of Translation
* 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
34
Ribosome sites
* 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
Translation Elongation
* 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
Translational inhibiting antibiotics
* bacteriostatic drugs
* tetracycline blocks A
* streptomycin blocks fMet-tRNA
* chloramphenicol and erythromycin bind to 50S
37
Translational Termination
* Stop codons are used
* No tRNA with anticodon that matches the stop codons
* release factor enters the A site and the ribosome disassembles
38
Mutations
* 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
Single mutation impact on pathogenicity
* 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
protein secretion (5 types)
* 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
SRP mediated transertion steps
* 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
SRP general info
* 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
Sec mediated secretion
* 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
Twin Arginine Translocase
* 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
Type III Secretion System
* 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
Type I Secretion System
* 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
Transformation Brief Intro
* 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
Detailed Transformation Steps
* 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
About Transformation
* 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
Noncompetent cells
* Can become competent without a transformasome
* uses electroporation, chemical transformation, pili
51
Quorum sensing
* 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
Type IV Secretion System pili Transformation
* 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
Bacterial Conjugation Steps
* 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
Conjugation Info
* 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
General Transduction Steps
* 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
General Transduction Info
* 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
prophage
-phage DNA that is incorporated into the bacterial genome
58
Specialized Transduction Steps
* 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
Specialized Transduction Info
* 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
General vs Specialized transduction
* 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
Restriction modification steps
* 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
Restriction Modification Info
* 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
How to stop the lytic cycle
* degrade phage DNA before replication - restriction modification
* modify host range and avoid tail fibers
* mutate LPS to avoid receptors
* Mutate receptor
* CRISPR
64
CRISPERCas Steps
* 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
CRISPRCas info
* 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
CRISPR Application
* allows for site specific DNA editing
* disease
* increase ag production
* animal modification
* increase resistance to bacterial infection
67
Recombination Advantages
* 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
Newly acquired DNA fate
* 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
Generalized Recombination of 2 circular DNA
* 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
2 circular recombination steps
* 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
Generalized Recombination (linear and circular)
* 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
Mutation effects
* neutral - no change in phenotype
* gain - advantageous
* loss - deleterious
* knockout mutation - null mutation (no protein made)
73
Point Mutations
transition - purine to purine or pyrimidine to pyrimidine
transversion - purine to pyrimidine or pyrimidine to purine
purine (A, G)
pyrimidine (C, T)
74
Inversion mutation
* double strand break and reverse order
* wild type - most common, unmutated, original
75
Reversion Mutation
* change to mutation and then reverse to wild type
76
Nucleotide excision repair (NER) steps
* 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
Base Excision Repair
* 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
Quorum Sensing Steps
* 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
Quorum Sensing Info
* 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
Vibrio Cholerae
* 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
Two Component System
* 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
LacZYA operon components
* lac Z - B galactosidase - enzyme cleaves lactose
* lac Y - permease - transmembrane pore that imports lactose
* lac A - transacetylase - unknown function
83
About Lac Operon
* 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
Lactose and glucose levels chart
