Molecular motors Flashcards
3 Types of helicases?
DNA helicases (unwind duplex DNA to ssDNA, and process/move holliday junctions and d-loops)
**RNA helicases **(destabilise RNA structure, promote ribosome assembly, translation, RNA splicing)
RNA/DNA helicases (unwind RNA/DNA hybrids, can terminate transcription, regulate DNA replication initiation)
Basic properties of helicases?
Common, ubiquitous, essential. –>Associated with genetic disorders
Magnesium dependent.
Couple NTP hydrolysis with **directional translocation along nucleic acids **(and unwinding)
Normally dimers or hexamers.
Structure features of helicases?
(probably just Superfamily1)
All have RecA fold:
Which contains: single NTP binding site allosterically linked to a distinct (RNA, DNA or RNA/DNA) polynucleotide binding site
What is the difference between type A, type B and bipolar helicases?
Direction of translocation. Known as POLARITY
Type A: 3’ to 5’ direction
Type B: 5’ to 3’ direction (type b rhymes with 5 to 3)
Bipolar: 2 helicases that go the same direction together along both antiparallel strands.
What is a holliday junction and what do helicases like RuvAB (in ecoli) do to them?
A 4 stranded DNA junction created during homologous recombination in meiosis.
Branch migration, movement of holliday junction along strands. (powered by RuvAB)
3 Parameters of helicase function?
Rate: 10-2000 bp/sec (modulated heavily by other proteins)
**Processivity: **how many bp unwound in any 1 polynucleotide interaction. 10 - 30000+ (modulated by other proteins)
**Step size: **how many base pairs unwound per ATP hydrolysed (theoretical upper limit 6, practically 1-3bp per ATP)
Features of PcrA - a model superfamily 1 helicase?
Plasmid Copynumber Reduction A
Functions in plasmid rolling circle DNA replication and repair (in staph aureus)
ATPase, binds ssDNA, 3’-5’direction,slow rate and processivity, 1bp stepsize
(RepD massively increases processivity)
General methods of helicase translocation?
Inchworm or Active rolling
General methods of helicase polynucleotide unwinding?
Passive (thermal fraying)
Active (ATP-dependent duplex distortion)
What is AMP-PNP?
A structural mimic of ATP used in lab to elucidate structure of ATP bound proteins.
Characterististics of ssDNA translocation motor? (in PcrA helicase)
Aromatic stacking interactions between aromatic AAs and nucleobases.
ssDNA binding site (in highly conserved core region) conformationally changes in response to ATP hydrolysis.
How can helicase activity be modulated by partner proteins?
Activation
Loading
Catalytic modification
(of helicase)
Hexameric helicase mechanism of ssDNA translocation? (e.g. BPV-helicase)
Toroidal hexamer with 6 DNA binding loops in spiral staircase in central channel.
Each monomer has ATP binding pocket which changes status sequentially around ring, and allosterically alters ssDNA binding loop position.
This conveys ssDNA through channel with high processivity.
[so hexamer helicases often part of replisome]
Structure of microtubules?
Fibrous proteins consisting of protofilament polymers of alpha and beta tubulin heterodimers.
13 Protofilament tracks align in parallel to create hollow tubes.
Microtubules have positive and negative ends!
To which end of microtubules (plus or minus) do kinesin and dynein walk?
Kinesins to plus, positive end
Dyneins to minus end, negative end
Features of conventional kinesin, kinesin 1:
Light chain at c-terminus forms **globular cargo-binding domain **(binds membranes like vesicles or organelles)
Long coiled coil stalk links to globular** n-terminal motor/ATPase head domains** via power stroke neck linker region
How does kinesin walk?
Towards the positive, plus end in 16nm hand over hand steps between ß-tubulin monomers.
Each step moves cargo 8nm. And requires one ATP hydrolysis
What do kinesin and myosin motors share, and what is different?
They share the same G-protein superfamily NTPase motor head groups. (but with different track binding sites, big region for actin vs small loop for ß-tubulin)
Different: power stroke components, Myosin has more complicated converter domain and lever armcompared to simpleneck-linker in Kinesin
(both walk to plus end)
How is ATP hydrolysis coupled to kinesin G-protein superfamily head motor movement?
By switch I and II sensor.
Switch II acts as spring loaded gate, moving dependent on gamma phosphate (of ATP) presence.
Switch II’s small movements are transmitted to the neck-linker region by the RELAY HELIX.
What is the powerstroke of kinesin motor?
neck linker “docks” to core motor on leading head, throwing partner head forwards (this occurs after ATP binding to leading head)
Partner head ADP bound (allowing it to release and bind new ß-tubulin)
(contrasted to myosin where ATP binding causes head release, and ATP hydrolysis causes cocking of free head)
What stimulates the release of ADP from kinesin leading head domain? (allowing the binding of ATP for neck-linker docking powerstroke)
Binding of the microtubule polymer stimulates ADP release from the kinesin.
What 2 things determine directionality of motor movement?
1) Directionality of protofilament binding
2) directionality of neck-linker powerstroke
Cytoplasmic dynein structure?
(Terminals other other way around to kinesin)
Complicated N-terminal light chains bind variety of cargo. (also dimerise protein)
Coiled coil stalks attach to c-terminal ring AAA+ motors with microtubule binding sites
(move towards negative, minus end, weirdos)
Single cytoplasmic Dynein motor structure?
Single chain Hexameric AAA+ motor with MTBD (microtubule binding domain) and a linker domain running across face of AAA+ hexamer to N-terminal tail region (dimerising).
(ATP binding to AAA+ ring causes conformational change of relative position between linker and coiled coil leading to MTBD and release of microtubule)
