prokaryotic molecular biology Flashcards
prokaryotes features and genome organisation
bacteria + archaea
absence of membrane-bound organelles
DNA in nucleoid
most DNA in B form (like regular double helix)
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
supercoiling
in circular DNA
double helix has additional turns or turns removed
adds tension and torsional stress
negative supercoiling
twist opposite to RH turn of helix
positive supercoiling
twist same direction as turn of RH helix
torsional stress
by formation superhelices or alter no. base pairs per turn (change amount of twisting)
linking number
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
topoisomerases
enzymes that alter linking number of DNA molecule
type I and II
type 1 topoisomerase
break 1 strand, pass other strand through gap and seal break
L changed by +/- 1
reduce coils so looser and easier for transcription/translation
type 2 topoisomerase
breaks both strands, pass another part of helix through
L changed by +/- 2
removes stress and can replicate both strands at the same time
E.coli genome organisation
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)
nucleoid
(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
different prokaryote DNA
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
horizontal gene transfer
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
main replicative enzyme
other enzymes in replication
polymerase III
pol I removes RNA primers from Okazaki
ligase links fragments
replicon
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
E.coli replication features
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
E.coli replication process
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)
control of E.coli replication
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
E.coli replication termination
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)
why is E.coli termination regions further apart than B.subtilis
because makes DNA as fast as can so spread them out in case 1 fork slower
recombination
breaking and rejoining DNA to new combinations for diversity and DNA repair
homologous recombination (definition + process)
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
non-homologous recombination
lack sequence similarity
RecBCD
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
transposition (3 main points)
genes change position on a chromosome
transposable genetic elements
Tn3 family
Inversion sequences
transposable genetic elements
definition, classes
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
insertion sequences
small (<2kb) encode proteins for transposition bound by inverted repeats cause direct repeats at site of insertion insert anywhere or specific target sites
transposons
Tn3 family
bigger
encode phenotypic characteristic like drug resistance
Tn3 family structure
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)
Tn3 family replicative transposition
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
Tn3 family non-replicative transposition
conservative
Tn10 + other transposons
(leaves no copy behind, just 1 molecule)
inversion sequences
alter orientation within DNA
control gene expression
salmonella spp inversion
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)
DNA damage
change from normal nucleotide sequence and supercoiled double helical state from physical/chemical env. and errors in replication
single base changes
structural distortions
single base changes
no effect on transcription/replication
e.g. keto-enol tautomerisation, deamination of cytosine to uracil, U rather than T, chem mods
structural distortions
may impede transcription/replication
e.g. single strand breaks, covalent modification of bases, removal of base, inter/intra strand covalent bonds, thymine dimer formation
thymine dimer formation
intrastrand binding from UV so won’t divide properly
2 adjacent thymines on same strand covalently link in cyclobutaine structure/ 6-4 photoproduct
DNA repair (5)
direct mismatch excision tolerance retrieval
direct DNA repair (and e.g.)
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
mismatch DNA repair (and e.g.)
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
excision DNA repair (and e.g.)
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
tolerance DNA repair (and e.g.)
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
retrieval DNA repair (and e.g.)
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
AP site
apyrinic/apyrimidinic
SOS response
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
transcription overview
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
RNA Pol
complete holoenzyme is a2bb’ωσ
σ factor easily dissociate to form core enzyme
holoenzyme
enzyme with coenzyme
factors controlling gene expression
promotor recognition promoter strength alternative σ factors guanosine tetraphosphate (iinhibits RNA synth.) mRNA degradation RNA processing regulatory RNAs regulate transcription initiation
promoter recognition
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
conserved promoter sequence
TATAAT - 10 Pribnow box
TTGACA - 35
UP mutations
make promoter more similar to consensus
consensus sequence
a sequence of DNA having similar structure and function in different organisms.
DOWN mutation
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)
UP elements and their recognition
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
initiation
release of σ factor increases affinity of RNA Pol for non-promoter DNA, change of shape locks Pol to DNA
alternative σ factors
transcribe specific subsets of genes (heat shock, motility, nitrogen metabolism)
constitutive expression
some genes that are always expressed
most genes are not constitutive but they are regulated and controlled
operon
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)
what are 2 transcription mechanisms that control gene expression?
induction - switching genes on when required, by activators (apoinducers)
repression - switching genes off by reressors
regulatory proteins like repressors are activated by
small molecules called effectors
inducers
activate the activators like apoinducers so turn genes on
co-repressors
activate repressors or inactivate activators so turn genes off
regulon
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
global regulation
a single environmental factor causes regulation of many genes with diff metabolic functions like SOS response which has 30 genes
lac operon structure
polycistronic RNA with 3 protein coding genes
lacZ
lacY
lacA
and lac I, lac P, lac O, CAP site
polycistronic
encode more than one polypeptide separately within the same RNA molecule
describe the lac operon sugar metabolism process
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
lacZ
beta-galactosidase
cleaves b-galactosides into monosaccharides
lacY
beta-galactoside permease
transports b-galactosides into cell
lacA
beta-galactoside transacetylase
detoxify b-galactosides by acetylation
lacI
repressor gene, always expressed
182bp up from lacZ and p/o/CAP
lacP
promoter
where RNA Pol binds
lacO
operator
overlaps promoter
where lac repressor binds so blocks promoter
CAP site
where cAMP acceptor protein binds
how does CAP interact with RNA Pol
alpha subunit of Pol interacts with 7AA surface-exposed beta-turn of CAP
trans-acting elements
diffusable products regulate gene expression on genes distant from where transcribed
cis-acting elements
regulate genes on DNA encoded on
complementation (and E.coli example)
if 1 on 1 chromosome working then normal phenotype
lac mutations (experiment with merodiploid E.coli so 2 copies or genes)
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
cloning
insertion of a DNA fragment into a self-replicating element to copy/isolate a particular piece of DNA
process of cloning
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
why is cloning used?
to analyse mRNA transcripts
it’s made into cDNA and then its RNA is cloned so reverse transcription
vector types
phage insertion vectors replacement vectors fosmids/cosmids Ti plasmid BACs YACs
phage vector
produce large numbers and incorporate DNA into chromosomes
can replace non-essential genes with cloned DNA
insertion vectors
non essential DNA already removed at restriction site
DNA cloned in
replacement vectors
non essential DNA replaced with non-coding stuffer DNA which can then be replaced by DNA to be cloned
fosmids/cosmids
large plasmids that clone large fragments
based on F-plasmids with co-sites (to help package vector into phage) and lacZ and T7 promoter
Ti plasmid
found in pathogen, used to transfect plant cells (but take pathogenic bit out beforehand)
BACs
bacterial artificial chromosomes
very large fragments
based on F-plasmid, carry selectable markers and lacZ gene
YACs
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
what do eukaryotes use to regulate gene expression?
small non-coding RNA (ncRNA)
e.g. addiction cassette in plasmids
1 type of addiction cassette
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)
hok sok system
host killing system by the R1 plasmid in E.coli is an example of an addiction cassette
RyhB
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
positive interactions in microbial communities
toxic product of 1 organism may be substrate for another
e.g. cyanobacteria needs heterotrophic bacteria
Windogradsky columns
mixed soil/sediment/waater/carbon/nutrients and leave for years
colours form from bacterial growth, whole ecosystem forms
negative interactions
Ab production targets others, competition for substrates
Quorum sensing
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
S. aureus quorum sensing process and effects
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
Agr
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
biofilms
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
5 stages of biofilm formation
- initial attachment - flagella and type I pili
- irreversible attachment - LPS and type IV pili
- maturation I - microcolonies form, produce sticky alginate and repress flagella so no movement
- maturation II - quorum sensing
- dispersion - release planktonic cells (single) from biofilm
P. aeruginosa biofilm
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
Vibrio parahaemolyticus biofilm
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
synthetic cell biology
lecture 5
