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Molarcular men biology > Perdy is NOT Bae > Flashcards

Perdy is NOT Bae Flashcards

1
Q

(1) Form of DNA used in most of cells

A

B form (2 chains are in opposite orientation)

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2
Q

(1) 2 ways torsional stress is accommodated in supercoiling

A

1) Formation of superhelices

2) Altering number of base pairs per turn of helix

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3
Q

(1) What topoisomerase type 1 does?

A

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)

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4
Q

(1) What topoisomerase type 2 does?

A

Breaks both strands of the DNA, pass another part of the helix through the gap (change linking number by +-2)

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5
Q

(1) Function of DNA gyrase?

A

Creates -ve supercoils (using ATP), opens up strands

essential for DNA replication

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6
Q

(1) Prophages definition

A

Sequenced bacterial genomes harbour phage-like elements, implicated in pathogenesis

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7
Q

(1) Genomic islands definition

A

Horizontally acquired genomic regions that may have mutated to mask modes of transmission and integration.

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8
Q

(1) Main replicative enzyme

A

DNA Polymerase III

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9
Q

(1) Role of Polymerase I in DNA replication

A

Role in removal of RNA primers from Okazaki fragments

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10
Q

(1) Outline process of DNA replication

A

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

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11
Q

(1) What is a replicon

A

Basic unit of replication (a DNA molecule or sequence which has functional origin of replication)

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12
Q

(1) 3 main features of OriC replication origin

A
  • GATC present 14x
  • 13-nucleotide motifs
  • 9-nucleotide motifs (DnaA binding sites)
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13
Q

(1) How is initiation of replication controlled?

A
  • 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.
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14
Q

(1) How does termination of circular chromosomes work?

A
  • 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.
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15
Q

(1) Why are replication forks arrested in one direction?

A
  • 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
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16
Q

(1) Definition of recombination

A

Breaking and rejoining of DNA molecules in new combinations

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17
Q

(1) 6 steps of homologous recombination

A

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

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18
Q

(1) 5 components of RecBCD enzyme

A

1) ssDNA exonuclease (5’ to 3’ and vice versa)
2) ssDDNA endonuclease
3) dsDNA exonuclease
4) DNA-dependent ATPase
5) DNA helicase

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19
Q

(1) 2 activity alterations to RecBCD when it encounters a chi site

A

1) 3’ to 5’ exonuclease activity INHIBITED
2) 5’ to 3’ exonuclease activity STIMULATED
(Helicase activity unaffected)

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20
Q

(2) Examples of single base changes (exert no effect on replication, but are mutations that are inherited)`

A
  • Replication errors via keto-enol tautomerisation
  • Deamination of C to U
  • Incorporation of U instead of T in repli
  • Chemical mod of bases
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21
Q

(2) Examples of structural distortions (impeding transcription/repli)

A
  • Single strand breaks
  • Cov mod of bases (alkylation)
  • Removal of base
  • Inter/intrastrand cov bonds
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22
Q

(2) Def Direct repair and example

A

Reversal/simple removal of damage

e.g. Photolyase reparing UV intrastrand pyrimidine dimers

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23
Q

(2) Def Mismatch repair and example

A

Detection and repair of mismatched bases

e.g. Uracil DNA glycosidase, removes U and adds T

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24
Q

(2) Def Excision repair

A

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)

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25
Q

(2) Def Tolerance systems and example

A

Allow DNA replication to proceed through damaged regions of DNA
(e.g. Translesion synthesis polymerases able to synthesise DNA past damaged bases)

26
Q

(2) Retreval systems and example

A

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

27
Q

(2) Explain the mut system

A

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.

28
Q

(2) Def of SOS response

A

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

29
Q

(2) Def transposable genetic elements

A

-Specific DNA sequences that are able to transfer copies of themselves to other parts of DNA.

30
Q

(2) The 7 classes of of transposable elements in bacteria

A

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

31
Q

(2) Explain phase variation in Salmonella spp

A
  • 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
32
Q

(2) 4 stages of transcription

A

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)

33
Q

(2) Promoter sequence features

A

1) -10 seq TATAAT

2) -35 seq TTGACA

34
Q

(2) What are UP and DOWN mutations

A

UP mutations make promoter more similar to consensus

DOWN mutations make less similar consensus.

35
Q

(3) Thy inducer?

A

Activate activators or inactivate repressors

36
Q

(3) Thy Corepressors?

A

Activate repressors or inactivate activators

37
Q

(3) What is diauxic growth?

A

When glucose is used first, and alternative carbon source is used after

38
Q

(3) 3 structural genes of the lac operon

A

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)

39
Q

(3) 3 alternative regions of lac operon

A

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.

40
Q

(3) 2 ways lac operon is ACTIVATED BRO

A

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)

41
Q

(3) How Jacob and Monod found the lac operon model

A
  • 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
42
Q

(3) WAT is Trans-acting elements

A

Gene product diffuses to affect both molecules

43
Q

(3) WAT is Cis-acting elements

A

Mutated lacO has no gene product, only affects DNA molecule its on.
-No repression occurs on chromosome operon

44
Q

(3) WAT is gene complementation

A

Both operons are repressed BUT f’ plasmid functional lacY

-Chromosome’s functional lacYZ complement mutations so phenotype is normal

45
Q

(3) HOW TO CLONE

A

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)

46
Q

(3) Enzyme to convert RNA to cDNA

A

Reverse Transcriptase

47
Q

(3) Why phages good vectors?

A

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

48
Q

(3) 2 Types of lambda vectors

A

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

49
Q

(3) Thy Fosmids?

A

Large plasmids based on F plasmid with cos-sites, lacZ and a T7 promoter added

50
Q

(3) Ti plasmid uses?

A

Used to transfect plant cells

-Only specific protion of Ti plasmid is transferred into the plant cell and then integrated into plant genome

51
Q

(3) How to clone large fragments?

A

USe BACs/YACs: Bacterial/Yeast artificial chromosomes

52
Q

(3) 3 areas of a chromosome

A

Telomere
Centromere
Origin of replication

53
Q

(3) Example of expression controlled by non coding RNA molecules

A

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

54
Q

(4) 2 Types of microbial community

A

Positive interactions: Toxic product of one organism may be substrate for another
Negative interactions: Antibiotic production that targets other microbes or competition for substrates

55
Q

(4) Process of Quorum sensing

A

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)

56
Q

(4) Process of accessory regulator regulatory sysyem

A

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

57
Q

(4) What are biofilms?

A

Structured clusters of cells, enclosed in self-produced polymer matrix and attached to surface (allows pathogens survival in environment and in hosts)

58
Q

(4) Common characteristics of biofilms

A
  • 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
59
Q

(4) 5 stages of biofilms

A

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

60
Q

(4) 2 examples of genes in biofilms

A

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)

Molarcular men biology (8 decks)

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