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MCB - Molecular Bio > prokaryotic molecular biology > Flashcards

prokaryotic molecular biology Flashcards

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Biology 101 flashcards
1
Q

prokaryotes features and genome organisation

A

bacteria + archaea
absence of membrane-bound organelles

DNA in nucleoid

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2
Q

most DNA in B form (like regular double helix)

A

2 polynucleotide chains in opposite orientations (run in opposite directions)
regular RH double helix
2nm diameter
complete turn ever 3.4nm
10.5 base pairs per turn helix
flexible - in terms of base pairs per turn, 3D gelix, bends

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3
Q

supercoiling

A

in circular DNA
double helix has additional turns or turns removed
adds tension and torsional stress

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4
Q

negative supercoiling

A

twist opposite to RH turn of helix

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5
Q

positive supercoiling

A

twist same direction as turn of RH helix

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6
Q

torsional stress

A

by formation superhelices or alter no. base pairs per turn (change amount of twisting)

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7
Q

linking number

A

how tight the twist (L)

total no. times 2 strands of helix of closed molecule cross each other when constrained to lie in a plane

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8
Q

topoisomerases

A

enzymes that alter linking number of DNA molecule

type I and II

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9
Q

type 1 topoisomerase

A

break 1 strand, pass other strand through gap and seal break
L changed by +/- 1
reduce coils so looser and easier for transcription/translation

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10
Q

type 2 topoisomerase

A

breaks both strands, pass another part of helix through
L changed by +/- 2
removes stress and can replicate both strands at the same time

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11
Q

E.coli genome organisation

A

supercoiled loops radiate from central protein core

proteins in nucleoid: DNA gyrase, topoisomerase 1, 4 heat unstable (HU) proteins for packaging DNA
forms tetramer around DNA (like histones in eukaryotes)

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12
Q

nucleoid

A

(meaning nucleus-like) is an irregularly-shaped region within the cell of a prokaryote that contains genetic material, not surrounded by a nuclear membrane like eukaryotes

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13
Q

different prokaryote DNA

A

not all have circular DNA
multipartite - divided into 2 or more DNA molecules (or might be plasmid)
sizes vary (obligates have smaller genomes)
min no. genes is 200 but obligate have 120

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14
Q

horizontal gene transfer

A 
rapid changes important in evolution
prophages - genomes with phage-like elements linked to pathogenesis
genomic islands (GIs) often mutated so pathogenesis
transposable genetic elements can change position in genome
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15
Q

main replicative enzyme

other enzymes in replication

A

polymerase III

pol I removes RNA primers from Okazaki
ligase links fragments

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16
Q

replicon

A

molecule that replicates from a single origin of replication
basic unit of replication
single one for each genome
DNA molecule/sequence replicated at least once per cell division cycle

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17
Q

E.coli replication features

A

oriC single origin of replication (replicon)
bidirectional replication leads to theta (egg with line through) structure
DNA digested with restriction enzymes and ligated to plasmid lacking origin of replication

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18
Q

E.coli replication process

A

1) initiation - 20 monomers DnaA bind to 4/5 copies of 9bp sequence in RH 2/3 (2 thirds) of oriC, DNA bends and forms closed complex
2) 3 copies of AT-rich 13bp sequence on LH 1/3 (a third) of oriC –> melt so open complex and separate DNA strands
3) DnaB helicase loaded onto melted DNA w/ DnaC, ATP hydrolysed so DnaC released
4) DnaB unwinds DNA in both directions which requires SSB protein (protects ssDNA) and DNA gyrase
5) RNA pol III makes DNA, RNA primer on both strands (primase)

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19
Q

control of E.coli replication

A

Dam methylase - methylates adenine residues GATC in OriC - replication only initiated if all 14 methylated

hemi-methylated replication because new strand unmethylated and can’t replicate so them re-methylated 1/3 through cell cycle

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20
Q

E.coli replication termination

A

2 forks approach each other and fuse in terminus region opposite oriC
arrest forks from 1 direction (polar)
Tus protein stops fork and waits for other form, if no other fork then carries on to next fork (ensures terminate both same time)

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21
Q

why is E.coli termination regions further apart than B.subtilis

A

because makes DNA as fast as can so spread them out in case 1 fork slower

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22
Q

recombination

A

breaking and rejoining DNA to new combinations for diversity and DNA repair

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23
Q

homologous recombination (definition + process)

A

2 sequences have similarity

1) alignment - of homologous regions
2) cleavage - of 1 strand at chi sites (RecBCD endonuclease)
3) invasion - 1 cleaved strand invades other sequence of 2 strands, Holliday junction (RecA), joined
4) branch migration - slide along and increase heteroduplex (RuvAB) so spread branch
5) isomerization - DNA twist, junction cross and uncross, chi form
6) resolution - crossed strands cleaved (RuvC), end depends on horizontal or vertical cleavage

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24
Q

non-homologous recombination

A

lack sequence similarity

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25
Q

RecBCD

A

binds end of dsDNA and unwinds with helicase activity
degrades both ssDNA strands with dual5’ to 3’ and 3’ to 5’ exonuclease activity
encounters chi site - 3’ to 5’ exonuclease inhibited, 5’ to 3’ stimulated so degrades 5’ end not 3’
ssDNA 3’ end tail and RecA binds to this

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26
Q

transposition (3 main points)

A

genes change position on a chromosome

transposable genetic elements
Tn3 family
Inversion sequences

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27
Q

transposable genetic elements

definition, classes

A

sequences that can transfer copies of themselves to other parts/other DNA/other orientation

7 classes in bacteria: insertion+composite, Tn3, phages, inversion, Tn7, Gram +ve

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28
Q

insertion sequences

A 
small (<2kb)
encode proteins for transposition
bound by inverted repeats
cause direct repeats at site of insertion
insert anywhere or specific target sites
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29
Q

transposons

A

Tn3 family
bigger
encode phenotypic characteristic like drug resistance

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30
Q

Tn3 family structure

A

lecture 2 page 1 diagram (IR, tnpA, res, tnpR, Bla, IR)
38bp terminal repeats (IR)
encodes 3 proteins TnpA + TnpR + Bla (beta-lactamase=ampicillin resistance)
3 cis-acting elements
internal resolution site (IRS/res)

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31
Q

Tn3 family replicative transposition

A

formation of cointegrate - 2 transposon copies in direct repeat occur at junction of component molecules (transposase)

recombination between 2 res sites forms 2 molecules each with res site (resolvase)

lecture 2 page 1 diagram

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32
Q

Tn3 family non-replicative transposition

A

conservative
Tn10 + other transposons
(leaves no copy behind, just 1 molecule)

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33
Q

inversion sequences

A

alter orientation within DNA

control gene expression

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34
Q

salmonella spp inversion

A

2 types of flagellum change to avoid immune system
phase 1/2 H antigen determined by orientation of hin region (H-inversion)

hin region - bound by 2 IR, encodes invertase

H1 has own promoter and operator and separated from hin region

H2 is an operon with rep gene which suppresses H1 and promoter within hin

hin faces certain way for promoter to point towards H2 so rep repressed H1 (phase 2)
if towards H1 then p for H2 in wrong orientation so H1 expressed (phase 1)

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35
Q

DNA damage

A

change from normal nucleotide sequence and supercoiled double helical state from physical/chemical env. and errors in replication

single base changes
structural distortions

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36
Q

single base changes

A

no effect on transcription/replication

e.g. keto-enol tautomerisation, deamination of cytosine to uracil, U rather than T, chem mods

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37
Q

structural distortions

A

may impede transcription/replication

e.g. single strand breaks, covalent modification of bases, removal of base, inter/intra strand covalent bonds, thymine dimer formation

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38
Q

thymine dimer formation

A

intrastrand binding from UV so won’t divide properly

2 adjacent thymines on same strand covalently link in cyclobutaine structure/ 6-4 photoproduct

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39
Q

DNA repair (5)

A 
direct
mismatch
excision
tolerance
retrieval
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40
Q

direct DNA repair (and e.g.)

A

reversal/removal

photolyase: photoreactivation repairs UV-induced dimer to create 2 thymine residues again
deoxyribopyrimidine photolyase enzyme contains 2 chromophores which absorb light and splits cyclobutane structures

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41
Q

mismatch DNA repair (and e.g.)

A

detect and repair mismatched bases

Uracil DNA glycosylase: when U instead of T, removes U making AP site then AP endonuclease breaks phosphodiester backbone at site and DNA Pol 1 binds and lays new DNA, gap sealed by DNA ligase

mut system: Mut S recognise mismatch/insertion/deletions (indels) and binds
MutL stabilise complex
Mut S-Mut L activate Mut H which locates methyl group and nicks opposite because new strand more likely wrong
Mut U helicase II unwinds DNA from nick to mismatch
DNA Pol 1 degrades and replaces DNA, ligase seals

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42
Q

excision DNA repair (and e.g.)

A

large bit of DNA replaced by undamaged (knows damage is somewhere)

E.coli 3 modes: very short patch, short 20, long 1500-10000bps
uvrABC enzyme bind damage and incision on both sides
UvrD separates strands
DNA Pol 1 and ligase

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43
Q

tolerance DNA repair (and e.g.)

A

replication proceed through damage

replace and guess what goes next
low-fidelity (incomplete) DNA pols synthesise past damage (almost all Y-family)

E.coli: pol IV and V
humans: 5 pols, pol n (eta) bypass UV photoproduct, defective in xeroderma pigmentosum (variant of skin genetic disorder) so helps prevent UV cancer

44
Q

retrieval DNA repair (and e.g.)

A

recombination with other copies of DNA

daughter strand gap repair: takes good bit from other DNA copy to replace wrong, then rely on other repair like excision, not actually repair itself and only when severe damage

45
Q

AP site

A

apyrinic/apyrimidinic

46
Q

SOS response

A

E.coli activates DNA repair genes if severe damage

LexA protein represses expression of SOS operons (LexA binds to LexA box in promoter of genes)

RecA changes conformation to active to induce SOS, inactivates LexA
inhibits 3’-5’ editing in DNA Pol III allowing error-prone replication

sulA inhibits cell division

47
Q

transcription overview

A

template recognition - RNA pol bind at promoter
initiation - 9 internucleotide phosphodiester bonds, sigma factor release
elongation - addition of nucleotides
termination - dissociation of enzyme, RNA and DNA rho-dependent/independent

48
Q

RNA Pol

A

complete holoenzyme is a2bb’ωσ

σ factor easily dissociate to form core enzyme

49
Q

holoenzyme

A

enzyme with coenzyme

50
Q

factors controlling gene expression

A 
promotor recognition
promoter strength
alternative σ factors
guanosine tetraphosphate (iinhibits RNA synth.)
mRNA degradation
RNA processing
regulatory RNAs
regulate transcription initiation
51
Q

promoter recognition

A

core enzyme bind many sites in loose closed complex with 1hr dissociation half life

holoenzyme loosely bound in closed complex has dissociation half life of 1 second

holoenzyme tightly bound in open complex has dissociation half life of >1hr

so σ makes RNA pol specific for promoters, tight stays for long time and melts DNA to open it

52
Q

conserved promoter sequence

A

TATAAT - 10 Pribnow box

TTGACA - 35

53
Q

UP mutations

A

make promoter more similar to consensus

54
Q

consensus sequence

A

a sequence of DNA having similar structure and function in different organisms.

55
Q

DOWN mutation

A

make less similar to consensus
mutations in -35 region reduces rate of closed complex formation (initial promoter recognition)
in -10 region reduces rate of open complex formation (required for melting)

56
Q

UP elements and their recognition

A

AT rich region upstream of -35 region which affects promoter strength (affects rate of transcription initiation)

alpha subunits involved in UP element recognition: 2 independently folded domains (alpha-NTD and alpha-CTD)
cleaved a-CTD can still dimerize and bind DNA and interacts with DNA of UP element
RNA Pol without a-CTD assemble and transcribe but no enchanced activity from UP

57
Q

initiation

A

release of σ factor increases affinity of RNA Pol for non-promoter DNA, change of shape locks Pol to DNA

58
Q

alternative σ factors

A

transcribe specific subsets of genes (heat shock, motility, nitrogen metabolism)

59
Q

constitutive expression

A

some genes that are always expressed

most genes are not constitutive but they are regulated and controlled

60
Q

operon

A

some genes transcribed together with single promoter and is regulated as a single gene with a linked function

polycistronic mRNA (more than 1 gene per mRNA)

61
Q

what are 2 transcription mechanisms that control gene expression?

A

induction - switching genes on when required, by activators (apoinducers)

repression - switching genes off by reressors

62
Q

regulatory proteins like repressors are activated by

A

small molecules called effectors

63
Q

inducers

A

activate the activators like apoinducers so turn genes on

64
Q

co-repressors

A

activate repressors or inactivate activators so turn genes off

65
Q

regulon

A

group of associated (by physiological function) genes, that may not be in the same operon but are controlled by a single regulatory protein

e.g. PHO in E.coli

66
Q

global regulation

A

a single environmental factor causes regulation of many genes with diff metabolic functions like SOS response which has 30 genes

67
Q

lac operon structure

A

polycistronic RNA with 3 protein coding genes

lacZ
lacY
lacA

and lac I, lac P, lac O, CAP site

68
Q

polycistronic

A

encode more than one polypeptide separately within the same RNA molecule

69
Q

describe the lac operon sugar metabolism process

A

E.coli uses diauxic growth which is using 2 substrates in succession not together

if glucose is available it is used first and it represses lactose enzymes (catabolite repression)
in the lag phase when glucose runs out, it induces lactose enzymes

b-galactosidase enzyme makes allolactose (lactose isomer) which binds to each 4 subunits of repressor
this causes an allosteric shape change so it can’t find the operator
IPTG binds repressor and activates promoter

no glucose activates adenyl cyclase to convert ATP to cAMP which activates CAP protein which binds CAP site and recruits RNA Pol to promoter

70
Q

lacZ

A

beta-galactosidase

cleaves b-galactosides into monosaccharides

71
Q

lacY

A

beta-galactoside permease

transports b-galactosides into cell

72
Q

lacA

A

beta-galactoside transacetylase

detoxify b-galactosides by acetylation

73
Q

lacI

A

repressor gene, always expressed

182bp up from lacZ and p/o/CAP

74
Q

lacP

A

promoter

where RNA Pol binds

75
Q

lacO

A

operator
overlaps promoter
where lac repressor binds so blocks promoter

76
Q

CAP site

A

where cAMP acceptor protein binds

77
Q

how does CAP interact with RNA Pol

A

alpha subunit of Pol interacts with 7AA surface-exposed beta-turn of CAP

78
Q

trans-acting elements

A

diffusable products regulate gene expression on genes distant from where transcribed

79
Q

cis-acting elements

A

regulate genes on DNA encoded on

80
Q

complementation (and E.coli example)

A

if 1 on 1 chromosome working then normal phenotype

81
Q

lac mutations (experiment with merodiploid E.coli so 2 copies or genes)

A

functional proteins (Z,Y,A) are still transcribed because mutated genes complemented by other chromosome

lacI still transcribed when 1 chromosome has mutated gene but there are some dominant mutations as well that overpower the other chromosome so mean the non-functional protein is transcribed that interferes with ability to bind operator

lacO and lacP binding site protein mutations are not transcribed

82
Q

cloning

A

insertion of a DNA fragment into a self-replicating element to copy/isolate a particular piece of DNA

83
Q

process of cloning

A

1) vector and DNA are restricted to make sticky ends which bind together and the gap is sealed with DNA ligase to make a circular recombinant plasmid
2) make competent with stresses like chemicals so can import into cell

3) select for plasmid-containing cells with selectable marker like antibiotic resistance so only cells with plasmid survive
and a vector screenable marker to determine plasmid contains DNA and not just vector with plasmid

84
Q

why is cloning used?

A

to analyse mRNA transcripts

it’s made into cDNA and then its RNA is cloned so reverse transcription

85
Q

vector types

A 
phage
insertion vectors
replacement vectors
fosmids/cosmids
Ti plasmid
BACs
YACs
86
Q

phage vector

A

produce large numbers and incorporate DNA into chromosomes

can replace non-essential genes with cloned DNA

87
Q

insertion vectors

A

non essential DNA already removed at restriction site

DNA cloned in

88
Q

replacement vectors

A

non essential DNA replaced with non-coding stuffer DNA which can then be replaced by DNA to be cloned

89
Q

fosmids/cosmids

A

large plasmids that clone large fragments

based on F-plasmids with co-sites (to help package vector into phage) and lacZ and T7 promoter

90
Q

Ti plasmid

A

found in pathogen, used to transfect plant cells (but take pathogenic bit out beforehand)

91
Q

BACs

A

bacterial artificial chromosomes
very large fragments
based on F-plasmid, carry selectable markers and lacZ gene

92
Q

YACs

A

yeast artificial chromosomes
based on real yeast chromosomes, grow in yeast cells
must contain origins of replication, centromere, telomeres
telomeres are joined and circularised to be stable
carry 2 selectable and 1 screenable marker

transfected into mutant yeast which is trp1- on one arm and ura3+ on the other arm and must contain both arms for growth
the cloning site is within the sup4 gene

93
Q

what do eukaryotes use to regulate gene expression?

A

small non-coding RNA (ncRNA)

e.g. addiction cassette in plasmids

94
Q

1 type of addiction cassette

A

makes stable mRNA of lethal membrane protein and also makes an anti-sense ncRNA which is unstable and binds to the mRNA and prevents translation
plasmid with ncRNA can pass to daughter cell in replication and so repress lethal mRNA (if no plasmid then dies from lethal mRNA)

95
Q

hok sok system

A

host killing system by the R1 plasmid in E.coli is an example of an addiction cassette

96
Q

RyhB

A

ncRNA involved in controlling iron use in E.coli
E.coli has proteins that need iron but they are not essential so when iron is limited FUR (global regulator) stops repressing ryhB which binds mRNA or non essential iron proteins and degrades them so the iron need falls

97
Q

positive interactions in microbial communities

A

toxic product of 1 organism may be substrate for another

e.g. cyanobacteria needs heterotrophic bacteria

98
Q

Windogradsky columns

A

mixed soil/sediment/waater/carbon/nutrients and leave for years
colours form from bacterial growth, whole ecosystem forms

99
Q

negative interactions

A

Ab production targets others, competition for substrates

100
Q

Quorum sensing

A

similar organisms send chemical signals and molecules like autoinducers (in response to cell population density changes) and sense each other
once threshold of autoinducers is reached, expression and function is changed
can use to create biofilm

101
Q

S. aureus quorum sensing process and effects

A

forms biofilms in wounds and medical devices and quorum sensing drives the switch from biofilm to invasive with the Agr regulatory system

at low density AIP is low so AgrC is inactive so no RNA III and no response so biofilm forms

biofilm matures so high density and AIP is high so activates AgrC receptor and AgrA is phosphorylated so activates P3 and produces RNA III

RNA III is an mRNA of hld (delta-hemolysin) and a regulatory RNA so represses adhesins and induces elements that drive invasion e.g. delta-hemolysin

102
Q

Agr

A

accessory gene regulator
operon with 2 promoters
expresses AgrA/B/C/D

D - AIP (autoinducing peptide)
B - transmembrane, secretes mature AIP
C - AIP receptor
A - response protein activates P3 so RNA III

103
Q

biofilms

A

structured clusters of cells enclosed in self-producing polymer matrix (from exopolysaccharides, proteins, nucleic acids), attached to surface and hides from environment like the immune system
hard to desiccate, often anaerobic

80% of all microbial biomass is in biofilms

104
Q

5 stages of biofilm formation

A
  1. initial attachment - flagella and type I pili
  2. irreversible attachment - LPS and type IV pili
  3. maturation I - microcolonies form, produce sticky alginate and repress flagella so no movement
  4. maturation II - quorum sensing
  5. dispersion - release planktonic cells (single) from biofilm
105
Q

P. aeruginosa biofilm

A

twitching motility used for maturation into microcolonies (stick to each other and sputum but not surface) and move along surface
sigma factor 22 changes expression

106
Q

Vibrio parahaemolyticus biofilm

A

switches between 2 flagella systems and interference with flagella leads to secondary swarming motility so flagella senses surface and interaction with it causes it to move along surface

107
Q

synthetic cell biology

A

lecture 5

MCB - Molecular Bio (7 decks)

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