Perdy is NOT Bae Flashcards
(1) Form of DNA used in most of cells
B form (2 chains are in opposite orientation)
(1) 2 ways torsional stress is accommodated in supercoiling
1) Formation of superhelices
2) Altering number of base pairs per turn of helix
(1) What topoisomerase type 1 does?
Breaks one strand of DNA, pass other strand through the gap and seal the break (Linking number changed by +-1)
-Removes one -ve supercoil
(Maintains supercoiling DNA with DNA gyrase)
(1) What topoisomerase type 2 does?
Breaks both strands of the DNA, pass another part of the helix through the gap (change linking number by +-2)
(1) Function of DNA gyrase?
Creates -ve supercoils (using ATP), opens up strands
essential for DNA replication
(1) Prophages definition
Sequenced bacterial genomes harbour phage-like elements, implicated in pathogenesis
(1) Genomic islands definition
Horizontally acquired genomic regions that may have mutated to mask modes of transmission and integration.
(1) Main replicative enzyme
DNA Polymerase III
(1) Role of Polymerase I in DNA replication
Role in removal of RNA primers from Okazaki fragments
(1) Outline process of DNA replication
1) DNA Pol III synthesises new DNA and Okazaki fragment
2) DNA Pol III stops when it reaches the RNA primer
3) DNA Pol I continues synthesis
4) DNA ligase links the 2 DNA fragments
(1) What is a replicon
Basic unit of replication (a DNA molecule or sequence which has functional origin of replication)
(1) 3 main features of OriC replication origin
- GATC present 14x
- 13-nucleotide motifs
- 9-nucleotide motifs (DnaA binding sites)
(1) How is initiation of replication controlled?
- Dam methylase methylates adenine residues in GATC seqeuences
- All 14 GATCs in oriC are fully methylated in initiation
- Newly synthesised strands NOT methylated but old strand is, to differ the strands.
(1) How does termination of circular chromosomes work?
- The 2 replication forks from OriC move bi-directionally away and fuse within a region diametrically opposed to OriC.
- DNA terminators are polar and only arrest forks in one direction
- Termination is achieved when the forks meet in the correct termination point, enabled by the stalling by ter regions.
(1) Why are replication forks arrested in one direction?
- The ter site must bind a specific terminator protein Tus (terminus utilisation substance) in E.coli
- Fork arrest results from inhibition of helicase-mediated unwinding of DNA duplex at apex of fork
(1) Definition of recombination
Breaking and rejoining of DNA molecules in new combinations
(1) 6 steps of homologous recombination
1) Alignment
2) Cleavage: One strand of each duplex is cleaved (RecBCD endonuclease at chi sites)
3) Invasion: Holliday junction formed (via RecA) (not identical molecules, heteroduplex formed)
4) Branch migration: Increase heteroduplex region (via RuvAB)
5) Isomerisation: Strands of HJ cross and uncross
6) Resolution: 2 crossed strands of HJ cleaved by RuvC
OUTCOME:
-2 duplex molecules with region of heteroduplex
OR 2 recombinant duplex molecules with a region of heteroduplex
(1) 5 components of RecBCD enzyme
1) ssDNA exonuclease (5’ to 3’ and vice versa)
2) ssDDNA endonuclease
3) dsDNA exonuclease
4) DNA-dependent ATPase
5) DNA helicase
(1) 2 activity alterations to RecBCD when it encounters a chi site
1) 3’ to 5’ exonuclease activity INHIBITED
2) 5’ to 3’ exonuclease activity STIMULATED
(Helicase activity unaffected)
(2) Examples of single base changes (exert no effect on replication, but are mutations that are inherited)`
- Replication errors via keto-enol tautomerisation
- Deamination of C to U
- Incorporation of U instead of T in repli
- Chemical mod of bases
(2) Examples of structural distortions (impeding transcription/repli)
- Single strand breaks
- Cov mod of bases (alkylation)
- Removal of base
- Inter/intrastrand cov bonds
(2) Def Direct repair and example
Reversal/simple removal of damage
e.g. Photolyase reparing UV intrastrand pyrimidine dimers
(2) Def Mismatch repair and example
Detection and repair of mismatched bases
e.g. Uracil DNA glycosidase, removes U and adds T
(2) Def Excision repair
Recognition of damage followed by excision of a patch of DNA and replacement by undamaged DNA. (Types: Very short patch (bp), short patch (20bp), long patch (1500-10,000 bp)
(2) Def Tolerance systems and example
Allow DNA replication to proceed through damaged regions of DNA
(e.g. Translesion synthesis polymerases able to synthesise DNA past damaged bases)
(2) Retreval systems and example
Recombinational processes to repair damaged DNA
(e.g. Daughter strand gap repair, allows replication after damage and relies on other systems to do it) Lazy sod
(2) Explain the mut system
1) MutS recognises mismatches and binds
2) MutL stabilises complex
3) DAM methylase methylates GATC seq on old strand
4) MutS-MutL complex activates MutH that finds methyl group and nicks new strand opposite meth group
5) MutU unwinds DNA from nick (towards mismatch)
6) DNA Pol 1 degrades and replaces unwound DNA
7) DNA ligase seals single strand break.
(2) Def of SOS response
If there is severe DNA damage, a large number of unlinked genes are activated to save the cell.
-LexA repress SOS operons
-RecA induces SOS response (by inactivating LexA)
SOS response allows cell to survive massive DNA damage by allowing DNA repli but expense of DNA repli fidelity
(2) Def transposable genetic elements
-Specific DNA sequences that are able to transfer copies of themselves to other parts of DNA.
(2) The 7 classes of of transposable elements in bacteria
1) Insertion sequences
2) Composite transposons
3) TN3 family transposons (e.g. drug resistance) alos known as replicative transposition
4) Transposable phages
5) TN7 transposons
6) Inversion sequences
7) Gram +ve transposons
(2) Explain phase variation in Salmonella spp
- Can express 2 different flagellum
- Culture of phase 1 cells will produce phase 2 at low frequency
- Phase variation determined by orientation of hin region
- Hin genes codes an invertase that catalyses inversion
(2) 4 stages of transcription
1) Template recognition: Binding of RNA polymerase to DNA template at promoter
2) Initiation: Formation of first 9 phosdiester bond and release sigma-factor
3) Elongation: Addition of nucleotides of growing RNA.
4) Termination: Recognition of a signal that E should cease followed by dissociation of enzyme, RNA and DNA. (Rho-dependent or independent)
(2) Promoter sequence features
1) -10 seq TATAAT
2) -35 seq TTGACA
(2) What are UP and DOWN mutations
UP mutations make promoter more similar to consensus
DOWN mutations make less similar consensus.
(3) Thy inducer?
Activate activators or inactivate repressors
(3) Thy Corepressors?
Activate repressors or inactivate activators
(3) What is diauxic growth?
When glucose is used first, and alternative carbon source is used after
(3) 3 structural genes of the lac operon
1) lacZ: B-galactosidase (Cleaves B-galactosides to monosaccarides
2) lacY: B-galactosidase (Cytoplasmic membrane protein that facilitates into cell)
3) lacA: B-galactosidase transacetylase (Detoxify toxic B-galactosides by acetylation)
(3) 3 alternative regions of lac operon
1) Promoter: lacP at which RNA Pol binds
2) Operator: lacO where lac repressor binds
3) CAP site: cAMP acceptor proteins (CAP) and catabolite respression protein binds here.
(3) 2 ways lac operon is ACTIVATED BRO
1) Inactivation of lac repressor (lactose to allolactose and this thing binds to it)
2) CAP activation (absence of glucose) by cAMP (by adenyl cyclase)
(3) How Jacob and Monod found the lac operon model
- Used merodipliod (partially diploid) E.coli by inserting lac operon genes into F’ plasmid
- Some genes encoded for diffusable products that regulated gene expression on BOTH DNA molecules
- Regulatory proteins can affect both DNA strands at site distant to their genes. TRANS-acting elements
(3) WAT is Trans-acting elements
Gene product diffuses to affect both molecules
(3) WAT is Cis-acting elements
Mutated lacO has no gene product, only affects DNA molecule its on.
-No repression occurs on chromosome operon
(3) WAT is gene complementation
Both operons are repressed BUT f’ plasmid functional lacY
-Chromosome’s functional lacYZ complement mutations so phenotype is normal
(3) HOW TO CLONE
1) Plasmid transform E.coli
2) Selectable marker to select for clones of vector (e.g. antibiotic resistance
3) Bacteria grown
4) Use a screenable marker (e.g. x-galactose blue thingy)
(3) Enzyme to convert RNA to cDNA
Reverse Transcriptase
(3) Why phages good vectors?
1) Good at producing large numbers and incorporating DNA into chromosome
2) Can replace non-essential genes (doesn’t affect lytic cycle) with cloned DNA
(3) 2 Types of lambda vectors
Insertion vectors: Non-essential DNA removed already and DNA can be cloned into this site
Replacement vectors: Non-essential is replaced with non-coding stuffer DNA that can be replaced by DNA to be cloned
(3) Thy Fosmids?
Large plasmids based on F plasmid with cos-sites, lacZ and a T7 promoter added
(3) Ti plasmid uses?
Used to transfect plant cells
-Only specific protion of Ti plasmid is transferred into the plant cell and then integrated into plant genome
(3) How to clone large fragments?
USe BACs/YACs: Bacterial/Yeast artificial chromosomes
(3) 3 areas of a chromosome
Telomere
Centromere
Origin of replication
(3) Example of expression controlled by non coding RNA molecules
IF IRON IS FREELY AVAILABLE
1) Protein FUR attaches to free iron, all-iron requiring proteins are made
IF IRON IS LIMITED
1) FUR stops repressing ryhB
2) RhyB ncRNA binds to mRNA of non-essential iron proteins which degrades
3) Need of iron of cell falls
4) Self regulates as iron concentration rises ryhB repression reoccurs
(4) 2 Types of microbial community
Positive interactions: Toxic product of one organism may be substrate for another
Negative interactions: Antibiotic production that targets other microbes or competition for substrates
(4) Process of Quorum sensing
1) Cells excrete a signal molecule (autoinducer) which allow them to sense population size
2) Once autoinducer reaches a threshold then cells respond (expression/function)
EXAMPLE
Staphylococcus Aureus can form biofilms on wounds but can switch to invasive phenotype driven by quorum sensing (via Agr system)
(4) Process of accessory regulator regulatory sysyem
Arg is 2 component reg system based around operon with 2 promoter
Agr D = autoinducing peptide (AIP)
Agr B = Transmembrane protein, secretes AIP
Agr C = AIP receptor, binds AIP then phos Agr
Agr A = Response protein, activates P3 producing RNAIII
Low cell density: Low AIP conc
High cell density (biofilm forms): AIP con rises
(4) What are biofilms?
Structured clusters of cells, enclosed in self-produced polymer matrix and attached to surface (allows pathogens survival in environment and in hosts)
(4) Common characteristics of biofilms
- Cells enclosed in polymer matrix of exopolysaccharides, proteins, nucleic acid
- Formation initiated by extracellular signals in environment
- Biofilm protects bacteria against the host immune response
(4) 5 stages of biofilms
1) Initial attachment: flagella, type I pili
2) Irreversible attachment: LPS, Type IV pili
3) Maturation I: microcolonies, repress flagella
4) Maturation II: Quorum sensing
5) Dispersion: release planktonic cells
(4) 2 examples of genes in biofilms
1) Pseudomonas aeruginosa (twitching motility essential for maturation into microcolonies = express type IV pili)
2) Vibrio parahaemolyticus (Switch between 2 flagella systems is important in maturation = interference with flagella rotation by sruface induces 2ndary flagella system linked to swarming motility)
