week 3 Flashcards
Hierarchy of regulatory mechanisms
DNA structure –presence, amplification, modification by porA/pili
Transcriptional regulation – activation,repression by sigma factor/fur
Post transcriptional regulation – translation repression, transcript stability by sRNA, riboswitch, RNAT
Gene
promoter, ORF, terminator
Operon
two or more genes connected all activated by a common promoter
Regulon
couple of genes alle regulated by the same regulator
Stimulon
genes activated by the same stimulus
Finetuning expression of genes why
Essential to adapt to growh conditions
Express virulence for pathogenesis and survival in host
function promotor, sigma, shine delgarno
Promotor is recognized by RNA polymerase and sigma factor, which recognizes -30 and -10
shine dalgarno in transcriptional region but not translational region AG rich is recognized by ribosome, recognizes AGG
DNA sequence alterations;
Slipped strand mispairing – DNA polymerase slips forward of back, leads illegitimate base pairing of repetitive DNA during replication and to insertion or deletion. Way to alter gene expression but very random. Caused by triplets or stretches of the same nucleotides
porA gene of n meningitidis
spacer becomes shorter, expression level of protein becomes less, because less binding strength RNA polymerase
transcriptional regulation
growth conditions
nutrients, oxygen, iron, glucose, pH
iron why needed in bacteria
needed to support growth, stimulus to produce new proteins used for survival (virulence factors). Could be potential vaccine candidates
identify iron regulated proteins in bacteria
RNAseq. Grow bacteria with and without iron, isolate cDNA, how many transcripts are produced can be measured. mRNA, RNA fragments, DNA framgents, map against sequence, reading map
transcriptional regulation
sigma factor
in contact with -10 and -35, equipped with RNA polymerase. Some sigma can bind specific sequences. Sigma 70 for genes that are always on and are general, sigma 32 for stress, sigmaE whe heatshocked, sigma 54 when nitrogen is present
mode of action of sigma factor
recognize sequence near promotor region, mode of action on repressor or activator protein
mode of action of repressor protein
steric hindrance (spacer region blocked for RNAP), blocking elongation, DNA looping
mode of action of activator protein
class I activation, class II activation, conformation change
sigma factor;
specific promotor sequence recognition
fur;
specific transcriptional regulator stimulated by Fe
A ferritine uptake regulator. Fur box is partly overlapping with promotor. Condition that there is a lot of iron present, fur will bind to fur box. No transcription is needed. Low iron – no binding fur to furbox, no transcription.
iron responsive regulon –
lactoferrin/transferrin binding protein
pili
* kapsel
* iron-acquisition proteins
* proteins complexed with iron (enzymes)
sRNA –
encoded in intergenic region or anti sense strand
regulate stability and translation mRNA, multiple mRNA targets
antisense to the 5UTR of target mRNA that they regulate
environmental stress induces – induces stress response
some contain fur box
mechanisms of riboregulation
translational repression – binding RBS
translation activation – remove pin like structure
mRNA degradation
mRNA stability
four regulatory mechanisms per sRNA
- Coupled degradation: both sRNA and mRNA degraded
- Sequestration: both sRNA and mRNA stabilization
- Catalytic degradation: only mRNA degraded
- Activation: with Qrr degradation
sRNA interaction with target
sRNA contains 4 stemloops that can disappear when bound to mRNA. When stemloop is intact, protein production is intact.
No loop after binding sRNA
RNAse can bind and degrade mRNA and sRNA
luxMN
degraded due to loss stemloop 1
luxO
not degraded due to stemloop 1 still being present
riboswitches –
elements in 5UTR of mRNA
operates by changing structure in response to binding metabolite
translation is on when RBS is fee, but when RBS binds to anti RBS then translation is blocked. Different ligands to block translation
RNATs
RNA thermometers
Also conformational change but due to temperature
RBS is in stemloop, when temperature raises RBS is free, ribosome can bind
Q fever symptoms
- 60% asymptomatic
- 20% fever like: headache, fever, nausea, muscle pain
- 20% serious; fever, chest pain, severe head ache, diarrhoea, vomiting, atypical pneumonia (not reacting to normal AB), hepatitis, pericarditis, meningo encephalitis
- 1-3% chronic (could be in any group); endocarditis and intravasculair infections. Increased risk in pregnant, vascular disease, intravascular patches. Leads to abortion, premature birth
Q fever cause
Coxiella burnetii
* Spores survive up to 40 months
* In rodents, birds, farming animals, cats, dogs, ticks. Mainly sheep, goats, cattle
* Found in dung, urine, milk etc, spread through dried excreta, air borne
* Very rarely contaminated food or ticks
Treatment; acute doxy 200mg 1dd 14d, chronic; combination therapy 18m
Diagnosis q fever
- First 2 weeks pcr on dna Coxiella
- Week 3 and later Ab detection phase 2 IgM IgG
- Chromic; IgG phase 1
How was origin of q fever found – abortus storm by goats
Q fever policy of blood transfusion
- Decided that it was not a threat
- However – PCR on hottest samples, serology on half of group (follow up after second transfusion). ELISA serological plot has shown that 1/10 had antibodies in affected area
- Could be transfused, however blood was needed so PCR tests were implemented
Why weren’t farms shut down earlier after thousands of notifications
because of the financial impact of shutting down farms.
Virulence –
- competition
- invasion
- Sensing
- Attachment
- Overturning host defenses
- Interference with host processes
Secretion system
membrane complex
Secretion systems in gram negative cell envelope
class 1-6 protein complexes
based upon order of discovery. Macromolecular structures present on surface; in part adaptation from pili, flagella, conjugation system
* One step – crosses both membranes, with help of chaperones and ATP
* Two step (2 and 5) – first inner membrane via sec/tat, periplasmic chaperones, no energy source at outer membrane
Crystallography
refraction of beam line when x ray shines on cristal
* Purified protein in large amount
* Prepare using precipitation (trial and error)
* Obtain diffraction pattern using x ray
* Determine structure from pattern
Crystallography not suitable for membrane proteins why
- hydrophobic surface
- aggregation rather than crystallization
- difficult to purify in large amounts out of membrane
- need to solubilize them using detergents
electron microscopy
electron beams reflected by electron dense material
* biological samples need staining with heavy metals
* cyro EM – freezing at low temp. prevents radiation damage, no staining needed
* suitable for bacterial cells, sliced samples, purified complexes with lower concentrations, less pure, image averaging improves resolution and samples can be tilted which gives 3d tomography
type III system
‘injection needle’ makes hole in eukaryotic cell, from cytosol bacteria to cytosol eukaryotic cell
* example; yersinia outer proteins (YOPS) secreted via type 3 modulate host cell processes
* intimate attachment and anti-apoptotic and anti-inflammatory signals
cryo-EM tomography of type III complex
- prepare vitrified cells
- perform tomography
- image with EM
- do averaging of identified complexed
- no purification
