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ORGANISATION AND CONTROL OF PROKARYOTIC GENOME (BINARY FISSION (process…
ORGANISATION AND CONTROL OF PROKARYOTIC GENOME
STRUCTURE (BACTERIAL GENOME)
prokaryotes structure (as a whole)
size
smaller than eukaryotic genome
0.5MB to 10MB (million base pairs)
lacks membrane bound organelles
contains bacterias's complete set of genetic material
BACTERIAL CHROMOSOME
DNA
associated with non-histone proteins ('naked')
single circular DNA
DNA is double stranded
found in nucleoid
no nuclear envelope
each bacteria contains 2 chromosomes
contains
one origin of replication
one because it's circular
genes organised into operons
several genes together under the control of a single promotor (ref. protein synthesis)
no introns
folded into loops of 100 000 base pairs
each loop is a loop domain
loops are bound to central protein scaffold
attached to cell membrane
loop domains supercoil independently
complexed with DNA binding proteins
supercoiling affect's genes ability to be expressed
more supercoiled means RNA polymerase has less access to promotor
PLASMID
DNA
smaller
1kb to 300kb (300 thousand vs 0.5million)
double stranded
circular
extra-chromosomal
contains
one origin of replication
undergoes DNA replication independently of chromosomal DNA (function)
genes that are not essential for
survival
and
reproduction
under
normal conditions
normally, bacterial chromosomes contain housekeeping genes - these need to be passed down in reproduction
contains genes coding for beneficial proteins under
stressful conditions
eg: genes for antibiotic resistance (R factor)
eg: F factor (conjugation)
BINARY FISSION
asexual reproduction of bacteria
forming 2
genetically identical
daughter cells
process
DNA attaches to cell membrane
at mesosome
folded invagination (infolding) of membrane
DNA replication occurs
starts at ori iC (origin of replication)
ori C is attached to mesosome
DNA gyrase removes supercoiling
ends at termination sequence opposite ori C
can only proceed when supercoiling is removed
bidirectional
uses each DNA to synthesise a complementary daughter strand via semi-conservative model
forms 2 DNA molecules attached to mesosome
eukaryotic DNA replication is the same as prokaryotic replication
replication for both goes outwards from ori C but lagging strand is made discontinuously in the direction to ori C
Cell growth
cell elongates
each circular DNA strand attached to cell membrane separates
cell membrane folds inwards between DNA molecules
(cell membrane growth)
forms a double later across long axis of cell
new cell wall layers secreted within membrane layers
(cell wall growth)
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attached to mesosome but moves apart
to either side of the mesosome
single colony: nutrient medium for growth of bacteria - conditions for growth met --> each 'dot' is one colony
arises from a single bacteria cell
VARIATION
due to spontaneous mutation
insertion
deletion
substitution
VERTICAL TRANSMISSION
mutation in parental DNA inherited by daughter cells
over many generations, results in accumulations of mutations
genetic recombination (gaining genetic material)
HORIZONTAL TRANSMISSION
TRANSFORMATION
uptake of a DNA molecule by the cell from
surrounding environment
incorporation of DNA into recipient chromosome (such that it's passed down to offspring - heritable)
foreign gene with
different allele
replaces original gene through genetic recombination
DNA found in environment due to
released from cells when cell death occurs
artificially introduced
alters
genotype due to different allele
alters phenotype
test
virulent S strain with gelatinous coat
coat prevents immune system from inhibiting proliferation of bacteria
bacteria infects (hence virulent)
non-virulent R strain without gelatinous coat
inject mice with
S strain
mouse dies
R strain
mouse lives
heat-killed S strain
mouse lives
R strain and heat-killed S strain
mouse dies
R strain transformed to produce gelatinous coat
R strain gained DNA from heat-killed S strain with allele coding for coat
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TRANSDUCTION
transfer of bacterial DNA from one cell to another through a
bacteriophage
premise: bacterial genes incorporated into phage capsid
due to mistakes made in virus life cycle
phage injects viral genome and bacterial genes into another bacterium
GENERALISED TRANSDUCTION
assembly in
lytic cycle
when new viruses are assembled
plasmids or
fragments of degraded DNA
packaged into new phage particles
some viral DNA is left behind
capsid has limited space (limited quantity of DNA)
resultant virus injects bacterial DNA into another bacterium when it infects a new host
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random fragment of bacterial DNA packaged
any segment of DNA transferred
SPECIALISED TRANSDUCTION
not all bacteriophages
only those who undergo lysogenic cycle - where viral DNA integrates with host chromosome
eg: lambda phage
process
during lysogenic phase (replication) phage viral DNA integrates in a specific point in bacterial chromosome (ref. viruses)
some bacterial DNA next to viral integration site excised along with viral DNA (beginning of lytic phase)
when viral DNA (prophage) is excised from bacterial chromosome (replication)
bacterial DNA injected into new bacterium when virus infects another bacterium
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specialized
as only bacterial DNA near prophage is removed
same gene always passed on
CONJUGATION
direct transfer of DNA between 2 bacterial cells in contact
who gives what
RECIPIENT of DNA: bacteria without F factor or R plasmid (female strain)
DONOR of DNA: bacteria with F plasmid or R plasmid (male strain)
F plasmid
segment of DNA in chromosome or plasmid
codes for production of sex pili
R plasmid
codes for enzymes giving antibiotic resistance
genes encoding sex pili
- CRITERIA for conjugation
process
sex pili acts as a grappling hook that draws bacteria together
forms cytoplasmic bridge between bacteria
allows DNA transfer
TDNA formed
DNA of F factor nicked at origin of transfer
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TDNA exported to recipient via cytoplasmic bridge
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combining DNA from 2 different bacterial cells into the genome of 1 cell type
observed (test)
2 E. coli mixed, with each unable to synthesise a different essential amino acid for growth
needs x amino acid but can't make it and medium doesn't have it so it can't grow
when mixed, colonies grow
indicates
uptake of genes
from the other E. coli strain to synthesise the amino acid it needs
a lot of colonies so can't be from mutation
GENE EXPRESSION AND REGULATION
gene expression: transcription and translation of a gene into a functional product
gene regulation: control of gene expression
prevents complete genome from being continuously active in transcription (prevent wastage)
allows prokaryotes to rapidly respond to environmental stimuli
inducers and repressors transmit signals so genes are turned on or off
as operons are induced or repressed
BACTERIAL GENE STRUCTURE
OPERONS
cluster of structural genes coding for specific products
cluster all codes for related functions
transcribed into single polycistronic mRNA
mRNA with genetic information for synthesis of multiple polypeptides
with start and stop codons at each coding region -
polypeptides aer separate
cluster under control of a single promoter
genes + promoter: transcription unit
operator
within promotor
between promotor and structural genes
function
controls access of RNA polymerase to genes
controls whether genes are expressed or not
benefits
genes coding for enzymes in metabolic pathway (related function) can be coordinately regulated and controlled
under control of single promoter and operator
regulation of gene expression
no wastage of energy and resources
only required enzymes synthesised
respond to changes in environment
lac operon responds to presence of lactose, glucose
trp operon responds to presence of tryptophan
regulatory gene
upstream of promoter (outside operon)
codes for repressor protein
regulates expression of structural genes
mechanism
binds to operator and blocks transcription
MECHANISM OF REGULATION
negative control
operator switched on
RNA polymerase can bind to promoter and transcription can occur
operator switched off
by repressor protein
prevents binding of RNA polymerase to promoter
prevents movement of RNA polymerase
structural genes not transcribed
no enzymes or proteins formed
transcriptional regulation by repressor protein
operon is switched off by active form of repressor protein
positive control
regulatory protein binds to DNA and increases rate of transcription
REGULATION OF GENE EXPRESSION MODELS
LAC OPERON
structural genes coding for enzymes involved in
metabolism of lactose
polycistronic mRNA that yields
lacZ
beta-galactosidase
breaks down lactose into glucose and galactose
lacY
lactose permease
transports lactose into bacterial cell
lacA
galactoside transcetylase
needed in lactose metabolism
CONTROL OF OPERON
NEGATIVE CONTROL (BY LAC REPRESSOR PROTEIN)
absence of lactose
regulator gene (lacL gene)
codes for
active
lac repressor molecule
dimer (2 polypeptides forming quaternary protein)
2 recognition sites
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repressor protein is bound to operator
blocks binding of RNA polymerase
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just upstream of lac operon
presence of lactose
lactose from growth medium enters cell through lactose permeate (from LacY)
regulator gene (lacL gene) codes for lac repressor molecule
allolactose binds to repressor molecule (allosteric site)
isomer of lactose formed in small amounts from lactose entering cell
changes 3D conformation of repressor
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because lac operon has minimal level of transcription
affects whether genes will be transcribed in the first place
can happen because enzymes are inducible
produced in response to presence of lactose (only produced when needed)
lac operon is inducible
POSITIVE CONTROL (BY GLUCOSE, cAMP, CAP)
affects whether genes are transcribed at a higher rate than usual
regulatory protein binds to DNA and increases rate of transcription
high glucose concentration
bacteria express genes for breakdown of glucose continually at faster rates (preferentially)
glucose is preferred
respiratory substrate
for bacteria
metabolised first before lactose
transport of glucose into bacterial cell inhibits adenyl cyclase
adenyl cyclase cannot convert ATP into cyclic AMP (cAMP)
cAMP level is low
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low glucose concentration
adenyl cyclase not inhibited
can convert ATP to cAMP
cAMP levels high
cAMP binds to CAP to form cAMP-CAP complex
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glucose repression
DUAL CONTROL
glucose must be low concentration and lactose high concentration for maximal expression of genes in lac operon (glucose low, lactose high)
glucose high, lactose low
no expression (repressor bound, CAP not bound)
glucose high, lactose high
minimal expression (operon off) (repressor not bound, CAP not bound)
glucose preferred respiratory substrate, used up first
time lag
between depletion of glucose and expression of lac operon
glucose low, lactose low
no expression (repressor bound, CAP bound)
TRP OPERON
repressible system of gene regulation
mechanism (negative control)
regulation of tryptophan biosynthesis (from series of reactions to form tryptophan)
high tryptophan level
tryptophan inhibits first enzyme in reaction pathway
inhibits enzyme 1 (so genes are still expressed but can't function)
tryptophan is co-repressor to turn off transcription
cell stops synthesising enzyme by blocking transcription of genes (genes not expressed)
binds to and activates repressor
repressor-tryptophan complex binds to operator
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low tryptophan level
repressor protein is inactive
not bound to operator region
promoter available to RNA polymerase for transcription
5 structural genes are expressed (forming enzymes catalysing biosynthesis)
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negative control because repressor is bound
because enzymes are repressible enzymes
synthesis can be down regulated by the presence of end product
feedback inhibition at gene level (repressible operon)
structure
5 structural genes
codes for 5 enzymes catalysing tryptophan biosynthesis
gene 3 --> enzyme 3
gene 2 --> enzyme 2
gene 1 --> enzyme 1
promoter
operator
regulatory gene (
far away from
operon) trpR
codes for repressor protein