Exam 2 Flashcards
1
Q
What percentage of total protein is ribosomal protein?
A
20%
2
Q
Copy numbers of rRNA per type of cell
A
Archaea: 1 copy
Prokaryotes: 8 copies
Mammals: 100s of copies
3
Q
What does increased gene dosage for rRNA increase?
A
ribosomal mRNA
NOT riboprotein
So riboproteins are involved in feedback inhibition of rmRNA
4
Q
S15
A
Binds and causes pseudoknot which blocks the ternary complex
Ribosomal protein autogenous translational repression
5
Q
What is rate limiting in ribosome synthesis?
A
Synthesis of rRNA
6
Q
Core Promoter Structure
A
Consensus at -10
Near consensus at -35
Spacing optimal 17 bp (actually 16 bp)
7
Q
UP Element
A
Upstream promoter
Third recognition element for RNAP
Enhancer?
8
Q
Three RNAP recognition elements
A
- -10
- -35
- UP elements
9
Q
Composition of P1
A
Fis sites - UP - -35 - -10
10
Q
Fis
A
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
11
Q
DksA
A
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
12
Q
DksA Mutant
A
Doesn’t shut down rRNA transcription in stationary, increasing in fresh medium, respond to aa starvation or show growth rate dependent regulation
13
Q
How does DksA impact ppGpp?
A
Increases apparent Km of ppGpp for RNAP
Increases impact on open complex lifetime
14
Q
How does DksA impact iNTP?
A
Increases concentration required for transcription
15
Q
BoxA
A
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
16
Q
Stringent Response
A
During amino acid starvation
Shutoff of RNA synthesis
Produced in idling reaction between ribosomes and ppGpp synthase
17
Q
Stringent Response is dependent on what?
A
Charged tRNAs, not AA pool
18
Q
RelA
A
ppGpp synthase
19
Q
Relaxed Response
A
RelA null allele
No ppGpp accumulation in response to AA starvation
In fact decrease in ppGpp levels
20
Q
spoT
A
reversible ppGpp synthase and hydrolase (to GDP)
21
Q
gpp
A
Guanosine pentaphosphate
ppGpp degradation
null allele = hyperproduction
22
Q
ppGpp
A
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
23
Q
ndk
A
Nucleoside diphosphate kinase
Creates precursor for ppGpp
24
Q
How does ppGpp stop transcription of rRNA?
A
Binds at promoter P1
25
How could ppGpp negatively regulate?
1. Open complex stability
2. Promoter clearance
3. Open complex formation
4. Pausing during elongation
5. Competition between ppGpp and NTP substrates
6. Base pairing with cytosines
26
When do and which promoters require higher concentrations of NTPs?
Initiation
| P1 promoters
27
What pieces of evidence support that NTP concentrations are not saturation for rRNA promoters in vivo?
1. RNAP mutants requiring higher concentrations of iNTPs
| 2. Promoter mutants that no longer needed high concentrations of NTPs
28
What is one way you can control rRNA promoters in vivo?
Control the concentrations of GTP and ATP
29
How do Bacillus subtilis promoters differ from E. coli?
1. Less dependence on UP elements and alpha CTD
2. No Fis
3. All promoters initiate with GTP
4. ppGpp works indirectly by decreasing GTP
30
ppGpp deficiency genotype
1. No growth w/o aa
2. Filament formation
3. Decreased survival
4. Decreased virulence
5. Decreased expression of sigma S genes
31
What is the largest group of metal resistance systems?
Efflux pumps
32
What are the two types of efflux pumps?
1. ATPases
| 2. Chemiosmotic cation/proton antiporters
33
4 generalizations about metal resistance
1. Highly specific
2. No general mechanism for all heavy metal ions
3. Resistance on plasmids in every group tested
4. Resistance is usually efflux pumping or enzymatic conversion
34
Where genetically are efflux pumps found?
On both plasmids and chromosomes
35
Which metal resistance systems are highly conserved in bacteria and which aren't?
Arsenic and mercury are conserved
| Cadmium is not (evolved 3 times)
36
What are the 3 evolutionary paths of cadmium resistance?
1. ATPases in G+ bacteria
2. Antiporters in G- bacteria
3. Metallothionein in cyanobacteria
37
Metal homeostasis
Metals are not free atoms in cell cytoplasm, they are bound to carriers
38
Thiol titration
Metals have natural affinity for sulfur.
| R group of cysteine readily binds metals, blocks thiol use for normal function
39
Metal chaperones
Metals can be associated with chaperones and delivered to efflux pumps or enzymes
40
Metal Uptake
Poorly understood
| Via import pumps that lack discriminatory activity?
41
Cd2+ ATPase motifsd
```
G+
Cd2+ binding
Aspartyl kinase
Phosphatase
Membrane channel
```
42
P-type ATPases
All contain aspartyl kinase and phosphatase
| Only transport ATPases that have a covalent phospho-protein intermediate
43
CadC
Binds to cad promoter/operator
| Inducibly regulated by divalent cations
44
Family of metal binding proteins
ArsR, SmtB, CadC
1. Respond to metals
2. Repressors
3. HTH with metal binding cys residues
45
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
46
Wilson's Disease
Copper overload
| Impacts liver
47
Cadmium resistance Alcaligenes
Czc: efflux pump (chemiosmotic divalent cation/proton antiporter)
Mutations give Zn resistance
G-
48
Czc system components
Cadmium resistance
1. CzcA: Basic inner membrane transport protein
2. CzcC: outermembrane protein
3. CzcB: membrane fusion proteins that bridges cell membrane
B C C B
A A
49
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
50
Levels of copper and Cop system response
Low: CopY is inactive
Medium: CopY is active
High: CopY is inactivated by CopZ
51
ars operons
```
Found in G+ and G-
Reduce As(V) to As(III), which is more toxic
```
52
Components of ars operon
```
ArsR: transcriptional repressor
ArsD: regulatory throttle protein (upper limit)
ArsA: membrane associated ATPase
ArsB: Arsenite transport protein
ArsC: Arsenate reductase
```
53
What two ars genes are missing from E. coli chromosomes and staphylococcal plasmids?
ArsA and ArsD
| ArsD has little impact
54
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
55
How do staphylococcal and gram negative arsenate reductases differ?
Staphylococcal derives reducing power from thioredoxin
| G- derive reducing power from glutaredoxin
56
What is one way that arsenate can diffuse out of cells?
Metal inactivation by volatilization
Methylation of As to volatile species
Methanobacterium
57
What is the most toxic metal in humans?
Mercury causes neurological and immunological dysfunction
58
What is the trend for mercury resistance across domains?
Bacteria: MIC=100uM
Archaea: MIC=300 nM
Eukarya: MIC=submicromolar
59
What is the mercury mechanism of toxicity?
Decreases RNA synthesis by blocking transcription
| Depletes mRNAs
60
mer operon Genes
```
MerR: Regulation (repression and activation)
MerP: periplasmic binding
MerT: Transport
MerA: Mercuric reductase
MerD: Regulation
```
61
merA
Mercuric reductase
| Hg2+ > Hg0
62
MerR
Unique positive acting activator protein that twists and bends the DNA region in the presence of Hg2+, allowing RNAP to bind
Bind at inverted repeats
Needed to help TBP and TFB
63
MerB
Organomercurial lyase that breaks carbon-mercury bond in toxic substrates suck as phenylmercury acetate
64
Narrow spectrum mer systems
Systems with MerA but not MerB
| G-
65
Broad spectrum mer systems
Systems with MerA and MerB
| Confer resistance to organomercurials
66
Baseball glove model
MerT and MerP
Bind Hg2+ by a pair of vicinal cysteins in merP, followed by passing Hg2+ from cysteine pair to cysteine pair in MerT and finally to MerA
67
Chemical toxicity of uranium
Greatest risk
Interacts with phosphate groups of DNA
Kidneys
68
How is uranium present in oxic environments?
Soluble salts of uranyl ion
| When reduced from U(VI) to U(IV) it immobilizes
69
Important uranium reducing Bacteria
Geobacter
Shewanella (MtrA/SO3300)
Terminal electron acceptor, cytochrome mediated
70
Mutations in uranium reducing cytochromes negatively impacts
Fe(III) reduction rates with acetate as e- donor
71
Nanowires
Geobacter
Interact with insoluble terminal electron acceptors through pili
Electrons flow from cell to electron acceptor
Implied by localization of precipitated UO2
Reduce Fe(III) or Mn(IV) for sure, uranium probably
72
What can cause uranium oxidation?
Nitrate
Interaction with Fe(III), generating U(VI) and Fe(II) (coupled to oxidation of Fe(II) by nitrogen oxides created by nitrate reduction in bacteria)
Humic substances, siderophores and bicarbonate
73
Argyria
Side effect of silver
| Irreversible discoloration of the skin resulting from subepithelial silver deposits
74
What metal can coat catheters?
Silver
75
Silver resistance genes
```
SilRS: sensor/responder
FilE: periplasmic Ag(I) binding protein
2 efflux pumps:
SilP: P-type ATPase
SilCBA: Chemiosmotic Ag(I)/H+ exchange system
```
76
silE
```
Binds Ag(I) between two His residues
Can bind 5 Ag
```
77
Metallothioneins
Only in Synechoccus cyanobacteria
Related to small, thiolate-rich metal binding proteins of animals
SmtA protein contains 9 cysteine residues that bind divalent cations in N-terminal and C-terminal clusters
78
Preference of cation binding in metallothioneins
1. Zn2+
2. Cd2+
3. Cu2+
79
SmtA
Uptake and efflux of Zn
| Regulated without connection to SmtB
80
5 Strategies of Metal Resistance
1. Enzymatic Conversion
2. Reduced sensitivity
3. Permeability barrier
4. Intra/extra-cellular binding
5. Efflux
81
ICP-MS
Inductively coupled plasma mass spec
| Metal based approach to identify metalloproteins on a genome wide scale
82
Terminator sequences functional component
RNA
83
Intrinsic terminators
Rho-independent
GC rich palindrome
Poly-T and poly-U form an RNA stem loop
84
Extrinsic terminators
Rho-dependent
85
Rho
Homohexamer donut-shaped
Facilitates dissociation of RNAP-DNA-mRNA complex
N-terminal RNA binding domain
C-terminal ATPase domain
Translocates by ATP hydrolysis, unwinding RNA-DNA hybrid
86
Rho-dependent terminator sites
1. RUT (rho-utilization site)
2. Termination sequence (pause RNAP)
Over 200 bp
87
Nonsense mediated polarity
Ability of nonsense mutations to reduce downstream gene expression
Only occurs in prokaryotic organisms
Coupled transcription and translation
88
NMP located near what tend to be more polar?
The 5' end of the gene (upstream)
89
In what bacteria is rho essential?
E. coli, Rhodobacter and Caulobacter
90
In what bacteria is rho nonessential?
Bacillus and Staphylococcus
91
Bicyclomycin
Antibiotic that inactivates rho
Experimental tool
Increase basal level of tryptophanase operon
Relieved polarity
92
tna operon
Tryptophan-induced relief from rho-mediation termination in leader region
Catalyze tryptophan from indole
Basal level is low
93
NusG
Bacterial protein that modulates elongation and termination through interaction with RNAP and rho
Bridges Rho and RNAP
HELPS RHO
94
NudA
Controls RNAP pausing and termination
Bacteria and Archaea
Anti-termination factor
Decreases rate of rho-dependent termination
95
Archaea and extrinsic termination
In Methanogens upstream sequence influenced susceptibility to downstream termination site
Analogous to rho and RUT sites
Halobacterial bop gene shows strong transcriptional polarity
NMP in S solfataricus
96
Sso mer operon NMP
merRHAI
Nonsense mutation at 12 codon of MerH
Mercury sensitive phenotype, same as MerA disruption mutant
Reintroduction of MerH elsewhere did not restore WT
MerH is important for downstream expression
97
Transcription terminator in Sso
```
At merHA junction
Two transcripts: HA and H
Thermometer
80°C: primary secondary structure is at MerHA junction
37°C: numerous secondary structures
```
98
Transcription Termination in Archaea
```
Polarity
NMP
Terminators
Rho is missing
NusA and NusG are present
RNAP is different
```
99
Domains of RNAP
Alpha: interacts with promoters
Beta: RNA synthesis
100
Sigma regions
1. Binds promoter when bound to RNAP
2. Binds -10 pribnow box
3. Binds -35 element
101
Sigma programming depends on
1. Affinity
2. Abundance
3. Interference
102
Sigma 32
rpoH
30°C > 42°C
Sigma 32 genes are turned on
Increased cellular concentration through enhanced synthesis and stabilization
103
rpoH RNA thermometer
Small region downstream is responsible for high levels of expression (+)
Large region is required for thermal regulation, repression at low temps (-)
104
Clp System
Degrades sigma S
ClpX: substrate binding
ClpP: proteolysis
RssB mediates recognition by binding both sigma S and ClpX
105
RssB
Undergoes N-terminal aspartate phosphorylation to activate sigma S binding
Acetyl phosphate decreases in starvation
106
Anti-sigma factors
Bind conserved regions
107
Phage T4 AsiA
Alt protein ADP ribosylates alpha subunit of RNAP and decreases -35 affinity
AsiA binds sigma-70 blocks 4.2- -35 binding
Mod protein overcomes by providing RNAP alternate contact
108
Sigma 28
```
Flagellar assembly
Class 3 genes are delayed
FlgM binds FliA until basal body is complete
Basal body secretes FlgM
Free FliA transcribes Class 3 genes
```
109
Archaeal transcription
TATA and TBP
TFB
RNAP
BRE
110
Eukaryotic Pol I
Nucleolus
rRNA
alpha-amanatin resistant
111
Eukaryotic Pol II
Nucleoplasm
hnRNA
Alpha amanatin sensitive
112
Eukaryotic Pol III
Nucleoplasm
tRNA
Variable in response to alpha amanatin
113
Eukaryotic Pol II Enzyme order
1. TFIID, TBP and TAFs
2. TFIIA (helps TFIID bind promoter)
3. TFIIB assigns direction and recruits RNAP
4. TFIIF and PolI
5. TFIIE (clearance and further melting)
6. TFIIH and TFIIJ (help create bubble)
114
Archaeal Polymerase
Pol II like
| 12 subunits which are orthologs of Pol II
115
Archaeal general transcription factors
1. TBP, no TAFs
2. TFIIB (TFB) directionality
3. TFIIS fidelity
4. TFIIE-alpha stimulation of promoters
116
TBP evolution
Encoded in metazoan genomes for differentiation
117
TIPS
TBP-interaction proteins
N terminal domain like TAF, stimulate transcription
ATP/GTP binding P loop, homology to helicase RuvB
Found in Archaea TAF like
118
3 Ways to Repress Transcription
1. Block RNAP
2. Prevent activation
3. Prevent elongation
119
Lactose is composed of
Galactose + Glucose
120
LacI
Formers tetramer
| Represses in absence of lactose
121
Lac genes
LacZ: B-galactosidase
LacY: permease
LacA: transacetylase
122
B-galactosidase
Converts lactose to allolactose
123
CAP binds
alpha-CTD of RNAP
| Cooperative binding with DNA looping
124
CAP impacts
Lactose and arabinose operons
125
How can repressors stop RNAP binding?
```
Binding a site that overlaps the binding site of RNAP
Lamba CI
LexA
p4
LacI
```
126
How does LacI impede RNAP?
Allows binding to promoter
| Allows short abortive transcripts, but inhibits promoter clearance
127
CytR
Binds to -70, flanked by cyclic AMP receptor proteins at -40 and -90
Doesn't overlap RNAP binding site
128
Low sequence specificity high abundance proteins
Block transcription at promoter by covering relatively large DNA regions at a nucleation site
p6 from phage phi29
DnaA (oligomerizes)
H-NS
129
Repressors that block transition for closed to open complex
Unable to open DNA strands at -10 region
Group I: bind sites that overlap with RNAP
Group II: bind sites that do not overlap with RNAP
130
Repressor inhibiting promoter clearance
1. RNAP is stalled at +6 to +12 when binding consensus element too tightly
p4 interacts with alpha-CTD
2. Promoter clearance is inhibited when repressor binds downstream from RNAP
LacI
131
Histidine operon
E. coli and S. typhimurium
132
Histidine biosynthesis
ATP + PRPP > IGP > Histidinol phosphate > histidinol > histidine
40 molecules of ATP per histidine
133
What are the three ways histidine biosynthesis is regulated?
1. Allosteric control of enzymes
2. Attenuation
3. ppGpp
134
HisG
ATP phosphoribosyl transferase (first step) is feedback inhibited by histidine
Homohexamer
Binding and inhibition are cooperative
AMP and ADP inhibit
135
His attenuation needs
1. presence of his residues in leader sequence
2. mutually exclusive secondary structures
3. RP pausing
136
What secondary structure terminates his operon?
EF and poly U terminate (caused by a lack of stalling in leader peptide)
137
His antiterminator
Anything that prevents EF
| DE is physiological antiterminator (caused by stalling)
138
Histidine excess
EF termination structure forms
139
Histidine Limitation
No attenuator forms
| DE antiterminator
140
Histidine superattenuation
AB, CD and EF
| Upstream stalling
141
His Unlinked Mutations
1. hisR: tRNAhis mutants in hisR promoter - less charged tRNA - stimulates starvation - antiterminator
2. hisS: histidyl tRNA synthase
3. hisT: pseudourine synthase, decrease function of tRNA
4. hisW: gyrA decreased gyrase activity, decrease strength of hisR promoter
5. hisU: ribozyme that results in decrease tRNAhis
142
Tryptophan operon repression
Low: no repression, dimer repressor cannot bind
High: repression, dimer binds
143
Tryptophan attenuation
Leader sequence has trp encoded
Starvation - stall - 2 and 3 form, no termination
Abundance - no stall - 1 and 2, 3 and 4 form, terminator hair pin
144
pyrC
UMP > CTP in pyrimidine nucleotide biosynthesis
High CTP: pyrimidine excess, SD is obscured dur to mRNA binding
Low CTP: no mRNA binding, SD is available to ribosome
145
TRAP
G+, Bacillus subtilis
Ring-shaped multimer of 11 subunits
binds RNA segments containing NAG repeats
Gene expression decreases, overlaps antiterminator sequence
Shine Delgarno is hidden in hairpin
146
AT
Anti-TRAP protein
deficiency of charged tRNATrp
Gene expression increases
147
Tryptophan synthase genes
TrpA and B
148
T box mechanism
Uncharged tRNA pairs with leader RNA, favoring formation of RNA antiterminator structure
149
rtpA
AT
Lacks tryptophan - insensitive synthesis
Leader has tRNATrp sensing
150
rtp operon response to tRNA Trp
Charged: Transcription stops due to terminator loop
Mild charge: transcription proceeds, but RNAP inhibits rtpA translation
Severe charge deficiency: Ribosome stalls and doesn't block translation
