Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Prokaryotic Physiology > Exam 3 > Flashcards

Exam 3 Flashcards

1
Q

Components of the Sec complex

A

SecYEG = channel, SecDF and YajC = membrane accessory proteins for the channel
SP1 and SP2 = signal peptidases that cleave signal peptides off preprotein
SecA = motor protein that pushes preprotein through channel
SecB = chaperone protein that brings preprotein from ribosome to SecA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Type I Signal Peptide

A

Positively charged hydrophilic n terminus (n region), hydrophobic core (h region), neutral hydrophilic c terminus (c region)
AXA motif marks cleavage site
Proline and glycine at regional boundaries to prevent helical structure (better able to be recognized)
Recognized and cleaved by SPase I

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What types of proteins have their signal peptides cleaved by SPase II?

A

Lipoproteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

YidC

A

Helps to insert membrane-targeted proteins into membrane during secretory pathway, thought to be via a “gate opening” mechanism in SecYEG channel
Essential protein; G+ bacteria have two copies (YidC1 and2)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How to tell from an amino acid chain that a protein is bound for the membrane?

A

Alternating hydrophilic and hydrophobic sequences

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Cotranslational secretion mechanism

A

Signal recognition particle binds signal peptide, then either binds and activates cytoplasmic FtsY receptor or travels to membrane to connect with self-activated membrane FtsY, carrying protein and ribosome (ribosome nascent chain complex) over to SecYEG for protein translocation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Protein expression on G+ cell wall

A

Sortase enzyme cleaves preprotein at LPXTG motif (between T and G) and covalently bonds to Threonine
Lipid II uses pentaglycine crossbridge to nucleophilically attack the sortase-protein C-T bond and protein becomes attached to lipid II
Lipid II gets converted to PG during cell wall synthesis mechanism

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Examples of proteins expressed on G+ cell wall

A

Protein A - binds antibodies to prevent optimization and limit phagocytosis (eg in staph aureus)
Clumping factor A - binds host fibrinogen to promote bacterial adhesion
Various pili proteins like SpaA pilus
Spore forming proteins (e.g. in bacillus anthracis)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Why might you artificially add or remove a signal peptide?

A

Target protein to a less complex compartment for easier purification so you can obtain a sample of purified protein for studying

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Type I secretion system

A

Inner membrane bound ABC transporter and Outer membrane bound OMP channel connected by membrane fusion protein (MFP) that spans the periplasm
Substrates are large and often associated with virulence (eg HlyA hemolysin), and contain a Cterminal secretion signal and GGXGXDXUX sequence (where U is a large hydrophobic amino acid)
Secretion is one step from cytoplasm to extracellular, and sec independent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Type II secretion system

A

Protein platform lodged in inner membrane, with attached pseudopilus that acts as a piston to push substrates thorough a channel in the outer membrane (made of oligomeric secretin and a tiny Pilotin)
Unfolded Substrate enters periplasm via Sec complex, is folded there, then enters type II system and is pushed though outer membrane channel by the pseudopilus

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Type III secretion system

A

Spans inner, outer membrane and plasma membrane of host cell
Chaperones target effector proteins through a sorting platform (cytoplasmic), basal body (periplasmic), and secretory needle apparatus(through outer membrane and extra cellular) to translocon which forms a a channel for entry into host cell
Proteins have signal sequences and are secreted unfolded

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Type IV Secretion System

A

Sec dependent OR independent, can be used to excrete proteins or DNA (conjugation)
Substrate either enters periplasm via Sec and then is extruded through OM channel and pilus, or is taken all the way through entire channel through IM, OM, and then through pilus to plasma membrane

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Type V Secretion System

A

Auto transportation by a special protein with an amino terminal domain that recognizes Sec complex, an internal alpha or “passenger” domain, and a carboxyl terminal beta or “helper” domain
Sec transport of protein into periplasm, then protein itself forms channel and pushes itself through
Helper domain inserts into outer membrane and forms beta barrel channel, then passenger domain passes through
Passenger is either cleaved from helper domain by extra cellular proteases or remains membrane bound by the helper domain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Type VI secretory system

A

Needle sheath with piston that ejects effector proteins into other bacterial cells (competition)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Type VII secretory system

A

Used for transport across cell wall
Multimolecular base complex in the inner membrane allows proteins to traverse into periplasm, and from there they travel via an undetermined mechanism across the peptidoglycan, arabinogalactan, and mycolic-acid-containing outer membrane

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Two components of the two component sensory system

A
  1. Sensor/receptor (eg histidine kinase, contains conserved histidine residue that becomes autophosphorylated upon stimulus binding)
  2. Effector/response regulator (cytoplasmic substrate for kinase, receiver domain contains an aspartate residue that is phosphorylated by histidine kinase, allowing operator domain to exert downstream effects)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Phosphorylation of the receiver domain in the response regulator leads to a conformational change in what domain?

A

alpha4-beta5-alpha5 domain (within the effector portion of the molecule)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Signal transduction for bacterial motility behaviour

A

Positive stimulus leads to activation of Kinase receptor and phosphorylation chain leading to counterclockwise rotation of flagella and a “run” behaviour

Negative stimulus leads to phosphorylation chain leading to a clockwise rotation of flagella and a “tumble” behaviour

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Pho P/Q regulon

A

Two component sensory system in salmonella
Upregulated by low Magnesium concentration (stimulus), also stimuli specific to macrophage inner environment, and anti microbial peptides
Supports intracellular survival in macrophages, resistance to antimicrobials, and Mg uptake
Also upregulates salmonella pathogenicity islands that code for type 3 secretion systems that promote intramacrophage survival and invasion of epithelial cells
Discovered by inserting GFP in salmonella cells, allowing them to enter macrophages, and assessing which bacteria cells survived in the macrophages by counting green glowing macrophages with flow cytometry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

VicR/K system

A

Two component regulatory system of Streptococcus mutans
Oxidative stress leads to a chain reaction including the production of sugars that then trigger biofilm formation
Reason why sugar causes cavities is that the sugar triggers biofilm formation by S mutans in the mouth which leads to dental plaque and eventually tooth decay
Discovery method: GFP reporter strain placed after VicRK promoter lead to increased fluorescence intensity under oxidative stress (indicating gene upregulated under oxidative stress), then knocked gene out and found that mutant was more sensitive to oxidative stress than wild type (indicating gene up regulation helps with protection against ox stress)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Cpx A/R system

A

Two component system of actinobacillus pleuropneumoniae
Detects envelope/membrane/periplasm stress (eg misfolded proteins, surface adhesion), and responds by activating genes for proteases and enzymes (breakdown misfolded proteins, build peptidoglycan to repair cell wall) and repressing genes for pili and flagella (expensive, need to conserve energy and resources)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

CovR/S System

A

Two component system of group A streptococci, an invasive pathogen
Activated genetic responses to adapt to host condition and promote colonization (capsule biogenesis, surface proteins like adhesions, secreted proteins like cytolysins and anti phagocytic factors, etc)
Discovery: microarray study of gene expression in wild type vs mutant - full genome on microchip, probe with RNA from WT and mutant and look for binding differences

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

AlgZ/R system

A

Two component system of pseudomonas aeruginosa
Coordinates expression of type IV pili and alginate
ChIP analysis to determine that it also upregulates mucR expression, increasing c-di-GMP synthesis, supporting biofilm formation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

ChIP analysis

A

Chromatin immunoprecipitation
Investigation of transcriptional regulators

Express protein of interest, allow it to bind DNA, add chemical to stabilize bond between protein and DNA, add antibody that recognizes protein, fragment DNA and remove DNA-protein complexes, add proteases to degrade proteins and isolate DNA, sequence DNA and identify genes regulated by the protein

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Microarray study

A

Put genome onto microchip, probe with RNA from both a wild type and a mutant organism, look for binding differences to determine differences in gene expression between the two organisms

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

GFP reporter study

A

Add gene for green fluorescent protein between the promoter and gene sequence for gene of interest

Cells that fluoresce green are expressing that gene

Could use facs to sort them based on gene expression

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

Knockout study

A

Knock out a gene, observe effect on phenotype

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Extracellular biofilm matrix composition

A

Proteins, polysaccharides, lipids, nuclei acids
Matrix specific proteins (eg RbmA) have fibronectin domains that mediate interactions with surface receptors
Robust, protease resistant amyloid-like fibres provide matrix scaffold (assembly is energy independent!)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

Phages in biofilm

A

Prevent new bacteria from joining/invading
Ability to kill bacteria depends on how long bacteria has been with biofilm (with time will get enveloped and receptors will no longer be exposed to phage)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

Strategies to limit biofilms

A

Matrix degrading enzymes

Immunotherapeutics (antibodies bind and sequester matrix stabilizing proteins, bind adhesins to block attachment)

Develop inhibitor molecules small enough to infiltrate the biofilm

Signalling pathway disrupters to prevent bacteria from communicating with each other

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

Quorum sensing

A

Bacteria produce auto-inducers

Critical concentration of autoinducers indicates large enough population of bacteria is present - phosphorelay signal cascade is initiated that affects gene expression, often followed by an additional TCS system with histidine kinase and RR components

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

Example V cholerae phosphorelay system for quorum sensing

A

Low conc autoinducer leads to production of sRNAs that block the action of HapR
HapR itself blocks regulators vps and aphA that regulate polysaccharide production, therefore polysaccharide production is allowed when autoinducer is low conc (fewer bacteria = need more polysaccharide to build matrix and form biofilm)

High conc autoinducer results in no sRNA production, allowing HapR to function and inhibit polysaccharide production (lots of bacteria = biofilm already formed, don’t need more polysaccharide material to be added)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

Bacterial genome shape

A

Circular

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

Experiment to determine replication pattern of DNA

A

Meselson and Stahl

Grew E. coli in media with 15N isotope rather than the more abundant 14N
Transferred E. coli with only 15N DNA moved to media with 14N
First replication: produced DNA sample with 100% intermediate weight (between 15N and 14N) - excluded conservative theory
Second replication: produced DNA sample with 50% intermediate weight and 50% 14N weight - excluded dispersive theory

Conclusion: semi conservative replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

Mechanisms contributing to low error rate in DNA replication

A

Discriminatory base selection by the polymerase that adds bases (10^-5 errors per base per round of replication)

Editing of misinserted bases by the 3’->5’ exonuclease associated with the polymerase (10^-2)

Removal of remaining mismatches by postreplicative DNA (10^-3)

Total error rate = 10^-10 errors per base per round of replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

DNA Pol adds nucleotides to which end of DNA strand?

A

5’

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

Direction of DNA synthesis

A

Begins at origin, proceeds bidirectionally, 3’ to 5’ prime meaning one direction will be continuous (leading strand) and the other direction will be in fragments (lagging strand)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

Role of primase (DnaG) in replication

A

An RNA polymerase; makes the RNA primer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

Role of polymerase III in DNA replication

A

“Workhouse” adds nucleotides (5’ to 3’) and proofreads (3’ to 5’)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

Role of DnaA in replication

A

Initiator; Binds origin (at A-T rich sequence) and begins the opening of the replication bubble and recruitment of other replication proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

Role of SSBs in DNA replication

A

Single stranded binding proteins, keep open complex open (strands separate)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

Role of polymerase I in DNA replication

A

Replaces RNA primer ribonucleotides with deoxyribonucleotides

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

Role of DNA ligase in DNA replication

A

Links Okazaki fragments together on lagging strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

Role of Helicase (DnaB) in DNA replication

A

Unwinds DNA ahead of the replication fork

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

Role of Tus in DNA replication

A

Binds termination sequence, blocks replisome until other one catches up, counters helicase action to help end DNA replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

Replisome

A

Complex of proteins that make up the replication fork in DNA replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

Role of HU in DNA replication

A

Histone-like protein that helps DnaA bind the origin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

General steps in DNA replication

A

Formation of open complex

Prepriming complex

Priming

Replication

Termination

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
50
Q

DNA replication in depth steps: formation of open complex and prepriming complex

A

DnaA binds specific sequences in oriC, with help from HU

DNA unwinds (ATP dependent)

SSBs bind to keep complex open

DnaB (helicase) binds to both ends of open complex

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
51
Q

DNA replication in depth steps: priming and synthesis

A

Primase binds and makes 12nt piece of RNA, allowing for addition of DNA nts
DNA Pol III adds DNA nts one at a time to 5’ end of chain (continuous extension on leading strand, in Okazaki fragments on lagging)
RNA primers are removed and replaced with DNA by DNA Pol I
Ligase joins Okazaki fragments together

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
52
Q

Who discovered Okazaki fragments?

A

Tsuneko and Reigi Okazaki (husband and wife)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
53
Q

Who discovered the DNA polymerases?

A

Arthur Kronberg discovered Pol I
His son Thomas discovered II and III
(His other son Roger studied RNA polymerases)
Paula De Lucia isolated the gene for Pol I, named Pol A after her

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
54
Q

DNA replication in depth steps: termination

A

Tus binds termination sequence (Ter)

Ter allows one way replication

Ter stops replisome, believed to counter helicase action to stop its movement

Other replisome catches up and dislodges Tus - replication complete

Daughter chromosomes end up “tangled” - resolved through decatenation by Topoisomerase IV and XerC/D

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
55
Q

Resolution of chromosome dimers after replication

A

During chromosome replication, homologous recombination between sister chromosomes can result in a chromosome dimer

FtsK lines up dif sites at the septum where XerC/D (a recombinase) and topoisomerase IV can resolve the chromosomes into two separate daughters

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
56
Q

DNA helix size and shape

A

10.5 bases per turn, right handed helix

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
57
Q

DNA supercoiling

A

Supercoiling = storing energy in DNA as tension (twists)
Superhelical tension can be used to aid processes (packaging, open complex formation, etc)
Positive supercoiling = overwinding
Negative supercoiling = underwinding

DNA gyrase introduces negative supercoils
Topoisomerase removes supercoils

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
58
Q

Topoisomerase action on DNA

A

Topo I removes supercoils by breaking and rejoining DNA backbone

Topo IV removes catenations to help resolve dimers of replicated chromosomes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
59
Q

Gene architecture

A 
Coding strand (resembles the mRNA strand except with T instead of U)
Template strand (strand that is read during transcription to form the mRNA)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
60
Q

RNA Polymerase subunits

A

Holoenzyme: 2 alpha subunits, 1 beta, one beta’, one omega and one sigma subunit

Core enzyme: holoenzyme without the sigma subunit (sigma helps to guide enzyme to initiation sequence, then leaves)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
61
Q

Housekeeping sigma factor

A

Sigma 70 - binds most initiation sequences

Alternate sigma factors have a specific sequence or small subset of sequences they recognize (more specialized)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
62
Q

Sigma 54

A

Holoenzyme with sigma 54 is Not independently competent for transcription - requires activator sequence NtrC at far upstream enhancer sequence which is activated via a TCS and loops over to the enzyme to activate it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
63
Q

Transcription promoter sequence

A

Two sequences at -10 and -35 (with space between)

Stronger the closer it is to consensus sequence (TTGACA-17-TATAAT)

More nt differences = weaker promoter

64
Q

Transcription initiation steps

A

RNA Polymerase Holoenzyme binds promoter (closed complex)
DNA unwinds to form open complex
Ribonucleotides begin being incorporated

65
Q

Transcription elongation steps

A

Sigma subunit dissociates

RNA nucleotides added by core enzyme

66
Q

Transcription termination steps

A

Factor independent:
- secondary structures in newly formed mRNA cause polymerase dissociation
Factor dependent:
- Rho proteins bind newly made mRNA and move up chain to Pol-DNA complex
- Pol reaches Rho dependent termination sequence and stalls
- Rho catches up and dissociates the polymerase using ATPase driven energy

67
Q

Operon

A

Cluster of genes encoding for proteins with related functions
One promoter used for transcription of all genes in the cluster into one long mRNA strand that will code for separate proteins
“Polycistronic mRNA”

68
Q

Ribosome

A

Made of RNA and proteins
Small subunit binds mRNA, large subunit provides enzymes needed to form peptide bonds
2 sites: A (aminoacyl) site = where new charges tRNA binds; P (peptidyl) site = where amino acid is joined to peptide chain via peptide bond and where empty tRNA leaves from after losing its mRNA

69
Q

Translation steps: initiation

A

Ribosome binds mRNA at Shine Delgarno sequence, recognized by 16sRNA in the small subunit (30s) of the ribosome
Ribosome scans until it finds start codon (AUG)
fMet tRNA binds start codon

70
Q

AARS

A

Aminoacyl-tRNA synthetase

Adds correct amino acid to tRNA

71
Q

Translation steps: translocation

A

Peptide bond is formed between tRNA in A site and peptidyl-tRNA in P site
Uncharged RNA leaves the P site, ribosome moves so next codon is in A site

72
Q

Translation steps: termination

A

Release factor binds stop codon and dislodges growing polypeptide chain

RF1 recognizes UAG and UAA
RF2 recognizes UGA and UAA

73
Q

Attenuation

A

Coupling transcription to nutrient supply (don’t make molecules that are already abundant, vice versa)
Common mode of regulation for amino acid synthesis

74
Q

Attenuation at the trp operon

A

High trp levels = ribosome moves quickly (not waiting for trp to be available), favours formation of RNA 3-4 terminator structure
Low trp levels = ribosome moves slowly (waiting for trp), favours formation of RNA 2-3 anti terminator structure

Also: repressor molecule can bind operator, but only when bound to trp. therefore high levels of trp lead to repression of the trp operon

75
Q

Jacques Manod

A

One of the founders of molecular biology
Figured out gene regulation of the lac operon and trp operon
Nobel laureate
Anti-nazi-occupation activist

76
Q

lac operon

A

Operon encoding enzymes that help with uptake and breakdown of environmental lactose
No lactose in environ: lacI repressor binds operator, preventing RNA Pol from binding promoter and initiating transcription of the enzymes
Lactose in environ: lacI repressor binds lactose, can’t bind operator, RNA Pol is able to bind promoter and initiate transcription

IPTG = artificial inducer (mimics effect of lactose but isn’t broken down by enzymes - continual activation of operon)

77
Q

Lac Z

A

B-galactosidase; cuts disaccharide lactose into glucose and galactose

78
Q

Lac Y

A

Permease; lets lactose into cell

79
Q

LacA

A

B-galactosidase transacetylase

80
Q

Control of flagella expression

A

flhC and flhD = early genes
Bind to promoter to turn on middle genes

Middle genes code basal body and hook, during construction flgM is bound to sigma28. When basal body and hook is complete, flgM leaves sigma factor and is the first molecule to be excreted through the structure.

Sigma 28 now free to activate late genes, which code for the filament

81
Q

Cis sRNA

A

Regulatory small RNA molecule adjacent to the gene they control (often in plasmids), they have extensive homology with gene target and are very specific

82
Q

trans sRNA

A

Regulatory small RNA molecule separate from the gene they control, they have limited homology with gene target and thus require the aid of Hfq protein to stabilize the RNA-RNA interaction

83
Q

Riboswitches

A

Secondary Structure of gene sequence itself regulates its transcription

Eg at certain temperature, strand changes structure to one that is more/less permissible to the action of RNA Pol

84
Q

Mechanisms for bacteria to destroy/block incoming foreign DNA

A
  • Restriction modification systems (eg methylate host DNA to mark it as “self”, incoming DNA will be unmethylated and marked for destruction
  • block phage attachment (eg through receptor mutation)
  • block phage infection (less well known mechanism(s))
  • CRISPR (insert copy of foreign DNA into own genome, recognize it in future and destroy fast)
85
Q

CRISPR locus

A

Interspaced repeats with DNA homologous to that of phages and plasmids between them

A few adjacent genes associated: CAS genes (code for enzymes that process the locus)

86
Q

CRISPR stages

A

Adaptation/immunization: new foreign sequence is acquired and put into the CRISPR locus
crBiogenesis: locus is transcribed and the Cas proteins process the crRNAs
Interference: crCRNAs guide an endonuclease to a target DNA that is then cleaved

87
Q

Importance of PAMs in CRISPR

A

Sequence next to target sequence that is NOT taken up into the CRISPR locus

Only when the crRNA-endonuclease complex sees the target sequence WITH the PAM sequence will they attack it, they won’t attack if I’m they see the sequence surrounded by the CRISPR repeats - protects the self genome that has had the foreign DNA added to it

88
Q

Types of CRISPR

A 
Three types (I, II, V)
Classified based on the way the crRNAs are processed
Most commonly discussed is type II, which uses the cas9 endonuclease
89
Q

Lytic cycle of phage

A

Injection of phage DNA into cell
Replication of DNA within cell, synthesis of new phage particles
Packaging of DNA into phage heads
Lysis of cell, phages find more cells to infect and repeat cycle

90
Q

Hershey chase phage experiment

A

Looking to see whether DNA or Protein was the genetic material being injected by phages into bacteria

Labeled phage protein capsule with sulfur 35, labeled phage DNA with phosphorus 32. Let phage infect bacteria, then separated phages from bacteria through centrifugation

Sulfur was detected in the phage sample (protein was not deposited) and phosphorus was detected in the bacteria sample (DNA was deposited)

Proved DNA was the genetic material

91
Q

Lysogenic cycle of phage

A

Phage infects cell
Phage DNA becomes incorporated into host genome
Cell divides, prophage DNA is passed on to daughter cells
Under stressful conditions, phage DNA is excises from the bacterial chromosome and enters the lytic cycle (phage DNA replicated and phage proteins are made, phages are assembled and then released upon cell lysis)

92
Q

Phage plaques

A

Zone of lysed bacteria in culture is a plaque

Lysogenic phage cycle will result in turbid plaques (bullseye shape due to Lysogenically infected bacteria still alive in the middle, surrounded by lysed bacteria around it)

93
Q

Prophage

A

Dormant phage within a host bacterium

No genes expressed except one: cl, which codes for a repressor
This repressor also prevents other phages from infecting the same bacterium, giving the bacterium the appearance of immunity (but really it is already infected)

94
Q

Lysogen

A

Host bacterium containing a dormant phage
“Immune” to further phage infection (superinfection) due to already being infected and repressor gene from phage dna being expressed

95
Q

Cl and Cro

A

Genes controlling phage life cycle

Cl predominates: repressor levels high, cro gene inactive, levels of cro protein low, lysogenic
Cro predominates: levels of cro protein high, cl gene inactive, levels of repressor low, lytic. Cro then triggers production of other phage genes needed to continue lytic growth

96
Q

Phage-infected bacteria response to DNA damage

A

RecA protease activated, which then cleaves the phage Repressor protein

Inhibition of cro and stimulation of repressor expression are interrupted

Increased level of cro leads to lysis

97
Q

Phage gene expression timing

A

Very early: only N (regulator of early genes) and cro are on

Early: N, cll (regulator of cl repressor and integrate synthesis), cro, replication and recombination genes are on

Late:

  • lytic: head, tail, and lysis genes on. Early genes off
  • lysogenic: cl (repressor) and int (integrase which allows chromosome to enter E. coli genome) are on, then once chromosome is integrated only cl is on
98
Q

Phage genome integration into bacterial chromosome

A

Phage DNA is circularized
AttP (phage site) and attB (bacteria site) recombine to form attL and attR half-sites at either end of prophage
Reaction catalyses by integrase, a phage encoded factor

99
Q

Phage genome excision from bacterial genome

A

AttL and attR sites reform attP and attB sites of the phage and bacterial sequences
Catalyzed by integrase and Xis, two phage-encoded factors

100
Q

Plasmid

A

Extrachromosomal DNA
Usually circular, but can be linear
Can encode factors that allow growth in new environments (eg antibiotic resistance and virulence)
Can be shared horizontally (within same generation)

101
Q

Colicins

A

Toxins encoded on plasmids used by bacteria to kill other bacteria that don’t have the same plasmid as them (plasmid that encodes the toxin also encodes a toxin neutralizer)
Usually kill by forming pores or entering bacteria and acting as nucleases

102
Q

High copy vs low copy plasmids

A

High copy: “relaxed” (made in abundance), small size, replication unlinked to cell division, random partitioning. Eg ColE1 plasmid (makes colicins that kill species related to E. coli)

Low copy: “stringent” (make few copies), large, replication linked to cell division, directed partitioning, often conjugative. Eg R100 plasmid

103
Q

Regulation of plasmid copy number in E. coli

A

RNA I = antisense RNA that binds RNA II, the promoter for plasmid replication

Rop = protein that helps with pairing of RNA I to RNA II

When plasmid number increases, so does level of Rop and RNA I —> they then act to stop plasmid production

104
Q

Regulation of R100 plasmid copy number by antisense RNA

A

RepA = required for initiation of replication at oriV
CopB = repressor of repA, encoded by copB gene
CopA = Gene that encodes RNA which is antisense to the RepA sequence
When plasmid enters cell there is no copB and little copA so RepA is expressed until plasmid copy number reached
When copy number reached, CopB and CopA repress expression of RepA and following plasmid gene

105
Q

Plasmid incompatibility of R100 plasmid

A

R100 can’t coexist with related plasmids because the copA regulatory genes are similar. Therefore they will repress the replication of the related plasmid

106
Q

Regulation of R100 plasmid replication by iterons

A

Iterons = repeated sequences of DNA
RepA binds iterons and “handcuffs” two plasmids together, preventing further replication
Can also handcuff related plasmids (more plasmid incompatibility)

107
Q

Plasmid incompatibility

A

Plasmids can regulate their own regulation, as well as that of related plasmids in the same cell
In these cases, only one of the plasmids will be inherited
These plasmids are in the same “incompatibility group”

108
Q

Factors causing instability of plasmids: Plasmid integrity

A

Plasmids often have insertion sequences or other recombination “hotspots” that allow for deletions or inversions
One gene may be intact but others are lost or have a changed orientation
Can cause problems, eg antitoxin genes are usually less stable than toxin genes, so if a plasmids antitoxin gene is lost, the bacterium will die from the toxin

109
Q

Factors causing plasmid instability: partitioning systems

A

ccdB-ccdA toxin-antitoxin system encoded on some low copy plasmids
If plasmid partitioning doesn’t go right, cells that don’t inherit the plasmid will die because they don’t have the gene coding for the antitoxin ccdA

Hok-sok toxin-antitoxin system: Hoi encodes toxic protein, sok encodes antisense RNA that suppresses hok translation. if plasmid is lost, Hok is made and cell dies

110
Q

Factors that cause instability of plasmids

A

Plasmid integrity
Partitioning of plasmids
Differential growth rates (plasmid encodes genes that are not being used, gets lost to decrease burden)

111
Q

Transformation

A

Uptake of “naked” DNA that is free in environment
Competence = ability of organism to take up DNA
DNA enters as a ssDNA molecule

Naturally competent organisms become competent in response to growth phase (late log phase) or diffusible factors from quorum sensing
Competent organisms may have proteins that bind preferred DNA sequences (eg Neisseria and Haemophilus)

112
Q

Lab methods to encourage transformation (DNA uptake) in less competent organisms

A

Treat with CaCl - pokes holes in membrane, encouraging DNA entry

If that doesn’t work:
Electroporation (shock in DNA using capacitor, more common)
Biolistic transformation (shoot DNA in on gold particles, less common)
113
Q

Self-transmissible plasmids vs mobilizable plasmids

A

Self transmissible plasmids encode the machinery necessary to initiate conjugation of themselves

Mobilizable: can be conjugated but require Tra functions supplied in trans

114
Q

Transconjugant

A

Plasmid recipient following successful conjugation

115
Q

Plasmid conjugation mechanism

A

Single strand break in donor plasmid at oriT via endonuclease action of Dtr “relaxase”, rolling circle replication, plasmid that is transferred is a copy of original

Donor plasmid contains transfer (tra) genes with Dtr and Mpf component. Donor forms pilus via expression of Mbf genes and brings cells in close contact, pilus forms bridge to transfer DNA

Non conjugating plasmids can also be transferred if it contains mob genes and non site (oriT), by utilizing the pilus formed by the other plasmid

116
Q

Argobacterium

A

Plant pathogen
Uses type IV secretion to transfer DNA from bacteria to plant host
Causes tree tumours!
Exploited mechanism to transfer DNA into fungi, malaria, etc

117
Q

Recombination frequency

A

Probability that recombination will occur at a DNA site
Fairly constant except for some hotspots
Increased homology between sequences increases chance of recombination
Greater distance between the two DNA sequences increases chance of recombination

118
Q

RecA

A

Mediates all bacterial homologous recombination events by pairing homologous DNA segments together so they can exchange strands

RecA monomer binds a single strand of DNA (activated by presence of ssDNA eg during replication - particularly stalled replication), more RecA are recruited to make a nucleo-protein filament, this filament then goes looking for homology to pair with and recombine

RecA mutant will not be able to perform homologous recombination

119
Q

F plasmid

A 
Conjugative plasmid (contains oriT and tra genes)
Can integrate into chromosome (contains IS 2 and two copies of IS 3)

Cells described as F+ or F- depending on if they have f plasmid or not

Chance for homologous recombination between plasmid and chromosome DNA

120
Q

Hfr strains

A

Bacteria with an F plasmid inserted in their chromosome that results in lots of homologous recombinations

Hfr = high frequency of recombination

121
Q

F’ plasmid

A

Imprecise excision of F plasmid from chromosome leads to inclusion of small piece of chromosome in the plasmid - now called an F’ plasmid

These plasmids are useful for constructing partial diploids (merodiploids)

122
Q

Hfr mapping

A

Mate Hfr strain with various markers, stop chromosome transfer at different times by shearing cells apart
Order and timing of gene transfer directly reflects the order of genes on the chromosome

123
Q

Insertion sequence

A

Sequence that codes transposase flanked by inverted repeats

Duplicate random target sequence and insert themselves into sequence between the two duplicates

124
Q

Transposons

A

Like insertion sequences but have selectable marker
Noncomposite transposon: transposon gene flanked by inverted repeats (eg tn3)
Composite transposon: transposon gene flanked by insertion sequences (eg tetracycline resistance gene)

125
Q

Regulation of Transposition

A

Unregulated Transposition would be lethal
Transposase binds poorly to methylated DNA - takes advantage of hemimethylated DNA during replication to perform Transposition then
Preferentially acts in cis (unstable and doesn’t accumulate to high levels, binds DNA as soon as it is made therefore more likely t bind it’s own site)

126
Q

Lydia and Delbruck Fluctuation Test

A

Determining answer to the question does environmental pressure cause mutations (Wallace’s theory) or does it simply select for existing mutations (Darwin’s theory)?
Exposed multiple cultures of bacteria to toxic phage and measured proportion of resistant mutants obtained each time
If proportion of mutants was the same every time, would support directed mutation theory; if number was variable, would support spontaneous mutation theory

Results supported spontaneous mutation theory

127
Q

Point mutation

A

Base substitution (replace one base with another)

128
Q

Silent mutation

A 
Codon change doesn’t result in amino acid change
Eg UUA (leu) to UUG (leu)
129
Q

Missense Mutation

A

Codon change results in one amino acid changing to another

Conserved change: amino acids are similar enough that protein function is unlikely to change (eg hydrophobic amino acid to another hydrophobic amino acid)

130
Q

Nonsense mutation

A

Codon changes from amino acid to stop codon, resulting in truncated protein

131
Q

Frameshift mutation

A

Addition or deletion of base(s) (not in multiples of 3) that changes reading frame (all following codons affected
Usually hit a stop codon soon after

132
Q

Null mutation

A

Full deletion of a gene

133
Q

Mutation reversion

A

Second mutation that reverses the first

Insertion mutations can be reverted by precise excision of inserted DNA, deletion mutations can’t be restored by “true reversion”

134
Q

Mutation notation

A

Gene is italicizes, protein first and last letter is capitalized

Insertions lacZ::Tn10
Null mutations: delta lacZ

(Pretend lacZ is italicizes in both of the above)

135
Q

Essential gene

A

Gene where null mutation (full deletion) is lethal to organism

Can be studied by isolating mutations that are dead under one condition and alive under another (eg temperature sensitive mutants will grow at permissive temperature and die at restrictive temperature)

136
Q

Polarity of mutations

A

Nonsense and insertion mutations are often polar (impact downstream genes as well)

Potential Reasons:
Insertional mutation inserts a transcriptional terminator
Mutation may disrupt translational coupling

137
Q

Pseudoreversion

A 
Intragenic suppression (mutation is suppressed)
Second mutation acquired that masks the effect of the first
Eg amino acid codon changed to stop codon then back to an amino acid codon but a different amino acid than original - it masked the effect of the stop codon without reverting back to original amino acid
138
Q

Suppression of nonsense mutation by tRNA mutation

A

Pseudoreversion
Amino acid changed to stop codon (mutation) but tRNA develops mutation that allows it to recognize the stop codon as an amino acid codon (suppression of mutation effect)

139
Q

Compensatory mutations

A

Pseudoreversion of a mutation whereby one mutation prevents/creates one function (eg pathway inhibition) but another mutation affects another function that masks the phenotype (eg activation of different pathway that performs similar function)

140
Q

Mutator strain

A

Strain of organism with mutations in error prevention mechanisms - have much higher mutation rates, useful for lab work

Insert plasmid into mutator strain, let it acquire mutations, bring it back out and have a library of gene mutations to choose from

141
Q

Methods of increasing mutation rate

A

Treat with ethidium bromide: intercalating agent that inserts between paired bases

Treat with NTG or EMS: alkylating agents, modify guanine to generate O6-methylguanine (polymerase pairs base with thymine instead of cytosine) GC becomes AT

Treat with nitrous acid: deaminates bases

Treat with UV irradiation: causes cross links between adjacent pyrimidines (thymine-thymine dimers) leads to frameshift (polymerase skips over base)

142
Q

Ames test

A

Method of determining if substance is a carcinogen (dna mutagen)

Plate His- salmonella typhimurium onto His- media, add suspected carcinogen, count frequency of revert ants (back mutations) that are now His+ (colonies that successfully grow)

143
Q

Bacterial response to DNA damage

A

Polymerase stalls, leaving ssDNA exposed
RecA binds ssDNA and is activated
Activated RecA cleaves LexA repressor which results in multi gene activation (excision repair of DNA mutation by Uvr system, activation of alternative Polymerase PolV or PolIV)

PolV and PolIV are error prone so don’t want to use it all the time but it is good because it is not so “perfect” that it will freak out and stall over a mutation like polIII does

144
Q

Mutations commonly caused by Pol IV and PolV

A

PolV - base substitution or error free (Ok)

PolIV - frameshift (bad!)

145
Q

Mut complex DNA repair

A

During DNA replication, DNA is briefly hemimethylated - MutH binds hemimethylated GATC sequences.
MutS and MutL complex bind mismatched DNA
DNA is threaded through complex in both directions until it reaches MutH protein bound at hemimethylated GATC sequences
MutH cleaves unmethylated DNA
Complex of DNA helicase II and exonucleases degrade unmethylated DNA towards the mismatch
Gap is filled by DNA Pol II and nick is sealed by DNA ligase

146
Q

Complementation

A

Restore phenotype by replacing damaged gene with a good copy
Usually done on a plasmid
Used in lab to demonstrate phenotype is due to mutation
Knock out: phenotype shows, knock back in: phenotype goes away

147
Q

Epistasis

A

Interaction of genes that are not alleles, in particular the suppression of the effect of one gene by another

148
Q

Forward genetics

A

Have a phenotype, need to figure out what gene(s) are involved

149
Q

Reverse genetics

A

Have a gene, need to figure out it’s function/effect(s)

150
Q

16s rRNA gene sequencing (amplicom sequencing)

A

Technique to identify organisms in a microbiome

Amplify gene for 16s rRNA with PCR, sequence it, compare to known sequences to identify the organism(s)

151
Q

Meta genomic analysis

A

Sequence all DNA in a sample

Way more data than 16s rRNA sequencing but tells you about more organisms than just bacteria and presence of enzymes (what organisms are doing)

152
Q

Complementation group

A

Different mutations of the same gene that affect same phenotype can’t complement each other (are in the same Complementation group)
However different mutations on different genes with same phenotype affected can complement each other (different complementation groups)

153
Q

How do you know when you have found all the genes involved in a process?

A

Classical genetics: isolation of same mutation(s) over and over until you can assume there are no other mutations involved (genetic screen is “saturated”)
Genomics and next gen sequencing platforms allow for much easier saturation

154
Q

How to introduce a transposon?

A

Use suicide plasmid - conjugative plasmid with R6K origin of replication (requires Pi protein for replication)
Pi protein is supplied in trans by a temperate phage
Plasmid contains transposon with antibiotic selection
Mate donor (where plasmid replication is permitted) to recipient (plasmid cannot replicate)
Select for antibiotic resistance due to the transposon hopping - these organisms will have added transposon

155
Q

Signature tagged mutagenesis

A

Mutants with unique DNA sequence - grown in a pool and DNA isolated from each
Apply selection pressure
Isolate DNA from pool of mutants that made it through, compare to previous DNA sample
Missing mutants: indicate that the gene they were mutated for was essential for survival within the environmental condition

156
Q

TnSeq

A

Transposon mutagenesis meets next generation sequencing

Sequence whole genome - know where all genes are
Sequence all mutants, compare to genome to find where the transposon went
Identify all essential genes in one screen

Pool of Tn mutants -> selection pressure -> pool of Th mutants (compare input and output pool to see which genes were required for selection pressure)

Prokaryotic Physiology (3 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions