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Molecular Motors Flashcards

1
Q

What is the structure of the T7 replisome?

A

gp5 DNA polymerase

gp4 (N) RNA polymerase and (C) helicase

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2
Q

How fast replication?

A

1000nt/sec

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3
Q

How may Mg ions are required for the replisome?

A

Mg for phosphate of incoming NTP

Mg for activation of 3’OH

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4
Q

What does thioredoxin do to the replisome?

A

Improves processivity from 15nt to 200nt

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5
Q

Does the replisome stay attached to DNA?

A

SSB dissociates and reassociates

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6
Q

How is thioredoxin bound to the replisome?

A

to the thumb domain of gp5

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7
Q

How many gp5 are bound to gp4?

A

Multiple in waiting

2 in processive

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8
Q

How is gp4 bound to DNA?

A

hexamer bound to lagging strand

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9
Q

What is the mechanism of the replisome?

A

replisome at fork
helicase splits duplex
leading strand synthesis
lagging strand synthesis using primase and loop back to gp5

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10
Q

Why is lagging strand synthesis slower?

A

Primase is 10x slower.
Activity halts helicase
Proposed loop release and reattachment at next okazaki fragment

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11
Q

What are the 2 modes of gp4?

A

Processive or waiting

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12
Q

How is fidelilty maintained by gp5?

A

NTP bound by finger domain

Incompatible transferred to exonuclease site by 3nm movement of thumb domain

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13
Q

Is thioredoxin always clamped to the replisome?

A

Yes, even in waiting mode

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14
Q

How is helicase movement driven?

A

ATP hydrolysis drives Brownian Rachet model with the new NTP acting as the pawl to drive forwards direction

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15
Q

What is the mechanism of the helicase?

A

molecular plough with canonical activity

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16
Q

How are helicases classified?

A

By substrate and direction

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17
Q

What functions do helicases have?

A

Protein removal
Remodelling of chromatin
Remodelling of protein:DNA complexes
Formation of RNA 2/3’ structure

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18
Q

When are helicases used?

A 
Transcription
Replication
Export
Splicing
Storage
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19
Q

What is the structure of monomeric helicases?

A

2 domains with central ATPase

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20
Q

How do monomeric helicases work?

A

Inchworm translocation mechanism. Hands are oppositely opened/closed by ATP binding

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21
Q

What protein family do helicases belong to?

A

P loop ATPases

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22
Q

What substrate do hexameric helicases most commonly have?

A

DNA

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23
Q

What is the mechanism of the low processivity RNA hexameric helicase?

A

ATP binding and hydrolysis propels RNA through channel

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24
Q

What is the mechanism of the high processivity RNA hexameric helicase?

A

ATP causes translocation of helix-loop-helix motif to bind RNA

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25
What is an example of the low processivity hexameric helicase?
Rho
26
What is an example of the high processivity helicase?
P4 packing
27
Why are proteases also molecular motors?
They use ATP to drive movement
28
What are the 4 classes of protease?
Serine Cysteine Aspartate Metallo
29
How specific is protease targetting?
Unspecific
30
Where are passive proteases?
Extracellular or compartmentalised
31
How can cytoplasmic proteases be regulated?
1-2nm gating | energy-dependant unfolding to feed into chamber
32
Where do proteases target?
Dynamic/exposed loop regions of compact proteins
33
What is an example of eukaryotic protease?
26S proteasome
34
Why are bacterial chambered proteases studied?
Simpler
35
Why are gated proteases uncommon?
Gating is smaller than many proteins
36
What are the 5 classes of bacterial chambered protease?
``` ClpAP ClpXP HsIUV FtsH Lon ```
37
What are the structure of the Clp and HsIUV proteases?
Hexameric AAA+ ATPases
38
What are the structure of FtsH and Lon?
Monomers with ATPase and protease | Combined into hexamer
39
What is a Clp?
caseinolytic protease
40
What is the Clp protease structure?
A/X unfolds P P A/X release
41
What is the structure of Clp P subunit?
2 heptameric rings, each with Serine Protease
42
What is the structure of Clp A subunit?
hexamer with 2AAA+ domains
43
What is the structure of the Clp X subunit?
Hexamer with 1AAA+ domain
44
How does ClpX select targets?
C/N terminal tags
45
How is the ssRA tag added?
On stalled ribosomes
46
How is the ssRA tag selected?
AANDENY- for SSpB adapter protein to link ClpX to substrate | -ALAA recognised by GYVG loop in ClpX pore
47
What are AAA+ ATPases?
ATPases Associated with cellular Activity
48
How big is the ATP binding domain of AAA+ ATPases?
200-250 residues
49
When are AAA+ ATPases monomeric?
ClpX in remodelling | ClpB in disaggregation
50
How can protease activity by observed?
Using tagged GFP and measuring decrease in fluorescence
51
Where do destabilising mutations have most degradative action?
Near tag
52
How much ATP is consumed during degradation?
1 for translocation, up to 500 for unfolding
53
How is degradation of ssRA tagged DHFR slowed?
By addition of methotrexate
54
How does force act as a vectorial denaturant in cells?
Motors, Signal transduction Switches Structural safety catches
55
What does atomic force microscopy measure?
Displacement of a spring of known stiffness to alter voltage in a photodiode receptor to produce a topology image
56
What strength does AFM measure?
25pN-nN
57
Comparison of laser traps to AFM?
Laser traps are a longer distance
58
What does lateral force microscopy measure?
resistance across a surface
59
What affects resolution of AFM?
Aspect of cantilever tip
60
What are the ideal AFM conditions?
Flat sample, flat surface, vacuum
61
What are the 2 modes of AFM?
Constant | Tapping
62
What is the advantage of tapping AFM?
Less damaging | More information from phase of oscillation
63
What resolution can be used for AFM of soft biological samples?
1nm
64
What can AFM be used for?
Low resolution structures Measurements of individual populations dynamics biomechanical properties

Proteins (12 decks)

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