Exam 2 Flashcards
What percentage of total protein is ribosomal protein?
20%
Copy numbers of rRNA per type of cell
Archaea: 1 copy
Prokaryotes: 8 copies
Mammals: 100s of copies
What does increased gene dosage for rRNA increase?
ribosomal mRNA
NOT riboprotein
So riboproteins are involved in feedback inhibition of rmRNA
S15
Binds and causes pseudoknot which blocks the ternary complex
Ribosomal protein autogenous translational repression
What is rate limiting in ribosome synthesis?
Synthesis of rRNA
Core Promoter Structure
Consensus at -10
Near consensus at -35
Spacing optimal 17 bp (actually 16 bp)
UP Element
Upstream promoter
Third recognition element for RNAP
Enhancer?
Three RNAP recognition elements
- -10
- -35
- UP elements
Composition of P1
Fis sites - UP - -35 - -10
Fis
Small DNA binding protein
Positive transcription factor for rRNA promoters
Binds at 3 sites in P1 and recruits RNAP
10x effect
Correlates to growth like ribosome concentration
Redundancy
DksA
Impacts cell division, sigma S, amino acid biosynthesis, quorum sensing and virulence
Transcription factor that binds RNAP
Essential for regulation of rRNA promoters through ppGpp and iNTPs
Reduces lifetimes of rRNA promoter open complexes
DksA Mutant
Doesn’t shut down rRNA transcription in stationary, increasing in fresh medium, respond to aa starvation or show growth rate dependent regulation
How does DksA impact ppGpp?
Increases apparent Km of ppGpp for RNAP
Increases impact on open complex lifetime
How does DksA impact iNTP?
Increases concentration required for transcription
BoxA
RNAP interacts with host factors and undergoes allosteric change that allows it read through rho-dependent terminators within rRNA genes
Termination at rho independent sites are not affected
Stringent Response
During amino acid starvation
Shutoff of RNA synthesis
Produced in idling reaction between ribosomes and ppGpp synthase
Stringent Response is dependent on what?
Charged tRNAs, not AA pool
RelA
ppGpp synthase
Relaxed Response
RelA null allele
No ppGpp accumulation in response to AA starvation
In fact decrease in ppGpp levels
spoT
reversible ppGpp synthase and hydrolase (to GDP)
gpp
Guanosine pentaphosphate
ppGpp degradation
null allele = hyperproduction
ppGpp
Magic spot
GTP+ATP
Down regulates DNA replication, fatty acids, cell wall, lipids, ribosomal proteins, elongation factors, stable RNA
Up regulates stress proteins, amino acid biosynthesis, proteolysis, glycolysis, survival genes, oxidative and osmotic stress genes
ndk
Nucleoside diphosphate kinase
Creates precursor for ppGpp
How does ppGpp stop transcription of rRNA?
Binds at promoter P1
How could ppGpp negatively regulate?
- Open complex stability
- Promoter clearance
- Open complex formation
- Pausing during elongation
- Competition between ppGpp and NTP substrates
- Base pairing with cytosines
When do and which promoters require higher concentrations of NTPs?
Initiation
P1 promoters
What pieces of evidence support that NTP concentrations are not saturation for rRNA promoters in vivo?
- RNAP mutants requiring higher concentrations of iNTPs
2. Promoter mutants that no longer needed high concentrations of NTPs
What is one way you can control rRNA promoters in vivo?
Control the concentrations of GTP and ATP
How do Bacillus subtilis promoters differ from E. coli?
- Less dependence on UP elements and alpha CTD
- No Fis
- All promoters initiate with GTP
- ppGpp works indirectly by decreasing GTP
ppGpp deficiency genotype
- No growth w/o aa
- Filament formation
- Decreased survival
- Decreased virulence
- Decreased expression of sigma S genes
What is the largest group of metal resistance systems?
Efflux pumps
What are the two types of efflux pumps?
- ATPases
2. Chemiosmotic cation/proton antiporters
4 generalizations about metal resistance
- Highly specific
- No general mechanism for all heavy metal ions
- Resistance on plasmids in every group tested
- Resistance is usually efflux pumping or enzymatic conversion
Where genetically are efflux pumps found?
On both plasmids and chromosomes
Which metal resistance systems are highly conserved in bacteria and which aren’t?
Arsenic and mercury are conserved
Cadmium is not (evolved 3 times)
What are the 3 evolutionary paths of cadmium resistance?
- ATPases in G+ bacteria
- Antiporters in G- bacteria
- Metallothionein in cyanobacteria
Metal homeostasis
Metals are not free atoms in cell cytoplasm, they are bound to carriers
Thiol titration
Metals have natural affinity for sulfur.
R group of cysteine readily binds metals, blocks thiol use for normal function
Metal chaperones
Metals can be associated with chaperones and delivered to efflux pumps or enzymes
Metal Uptake
Poorly understood
Via import pumps that lack discriminatory activity?
Cd2+ ATPase motifsd
G+ Cd2+ binding Aspartyl kinase Phosphatase Membrane channel
P-type ATPases
All contain aspartyl kinase and phosphatase
Only transport ATPases that have a covalent phospho-protein intermediate
CadC
Binds to cad promoter/operator
Inducibly regulated by divalent cations
Family of metal binding proteins
ArsR, SmtB, CadC
- Respond to metals
- Repressors
- HTH with metal binding cys residues
Menkes Syndrome
Encode P-type ATPase
Lethal X-linked disease of copper starvation
Accumulates in upper intestinal mucosa but doesn’t move into blood
Copper requiring proteins are nonfunctional including superoxide dismutase
Wilson’s Disease
Copper overload
Impacts liver
Cadmium resistance Alcaligenes
Czc: efflux pump (chemiosmotic divalent cation/proton antiporter)
Mutations give Zn resistance
G-
Czc system components
Cadmium resistance
- CzcA: Basic inner membrane transport protein
- CzcC: outermembrane protein
- CzcB: membrane fusion proteins that bridges cell membrane
B C C B
A A
Enterococcus cop system
In response to both copper starvation and excess
CopA: uptake ATPase
CopB: efflux ATPase
CopY: repressor activated by intracellular Cu+
CopZ: anti-repressor
Levels of copper and Cop system response
Low: CopY is inactive
Medium: CopY is active
High: CopY is inactivated by CopZ
ars operons
Found in G+ and G- Reduce As(V) to As(III), which is more toxic
Components of ars operon
ArsR: transcriptional repressor ArsD: regulatory throttle protein (upper limit) ArsA: membrane associated ATPase ArsB: Arsenite transport protein ArsC: Arsenate reductase
What two ars genes are missing from E. coli chromosomes and staphylococcal plasmids?
ArsA and ArsD
ArsD has little impact
What makes ArsA special?
Converts ArsB chemiosmotic complex into ATPase
Membrane transport that can switch between primary and secondary active transport is novel
Can transport antimonite
Homodimer - 4 ATP binding sites
How do staphylococcal and gram negative arsenate reductases differ?
Staphylococcal derives reducing power from thioredoxin
G- derive reducing power from glutaredoxin
What is one way that arsenate can diffuse out of cells?
Metal inactivation by volatilization
Methylation of As to volatile species
Methanobacterium
What is the most toxic metal in humans?
Mercury causes neurological and immunological dysfunction
What is the trend for mercury resistance across domains?
Bacteria: MIC=100uM
Archaea: MIC=300 nM
Eukarya: MIC=submicromolar
What is the mercury mechanism of toxicity?
Decreases RNA synthesis by blocking transcription
Depletes mRNAs
mer operon Genes
MerR: Regulation (repression and activation) MerP: periplasmic binding MerT: Transport MerA: Mercuric reductase MerD: Regulation