Molecular motors

Question 1

3 Types of helicases?

Answer 1

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)

Question 2

Basic properties of helicases?

Answer 2

Common, ubiquitous, essential. –>Associated with genetic disorders

Magnesium dependent.

Couple NTP hydrolysis with **directional translocation along nucleic acids **(and unwinding)

Normally dimers or hexamers.

Question 3

Structure features of helicases?

(probably just Superfamily1)

Answer 3

All have RecA fold:

Which contains: single NTP binding site allosterically linked to a distinct (RNA, DNA or RNA/DNA) polynucleotide binding site

Question 4

What is the difference between type A, type B and bipolar helicases?

Answer 4

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.

Question 5

What is a holliday junction and what do helicases like RuvAB (in ecoli) do to them?

Answer 5

A 4 stranded DNA junction created during homologous recombination in meiosis.

Branch migration, movement of holliday junction along strands. (powered by RuvAB)

Question 6

3 Parameters of helicase function?

Answer 6

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)

Question 7

Features of PcrA - a model superfamily 1 helicase?

Answer 7

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)

Question 8

General methods of helicase translocation?

Answer 8

Inchworm or Active rolling

Question 9

General methods of helicase polynucleotide unwinding?

Answer 9

Passive (thermal fraying)

Active (ATP-dependent duplex distortion)

Question 10

What is AMP-PNP?

Answer 10

A structural mimic of ATP used in lab to elucidate structure of ATP bound proteins.

Question 11

Characterististics of ssDNA translocation motor? (in PcrA helicase)

Answer 11

Aromatic stacking interactions between aromatic AAs and nucleobases.

ssDNA binding site (in highly conserved core region) conformationally changes in response to ATP hydrolysis.

Question 12

How can helicase activity be modulated by partner proteins?

Answer 12

Activation

Loading

Catalytic modification

(of helicase)

Question 13

Hexameric helicase mechanism of ssDNA translocation? (e.g. BPV-helicase)

Answer 13

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]

Question 14

Structure of microtubules?

Answer 14

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!

Question 15

To which end of microtubules (plus or minus) do kinesin and dynein walk?

Answer 15

Kinesins to plus, positive end

Dyneins to minus end, negative end

Question 16

Features of conventional kinesin, kinesin 1:

Answer 16

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

Question 17

How does kinesin walk?

Answer 17

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

Question 18

What do kinesin and myosin motors share, and what is different?

Answer 18

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)

Question 19

How is ATP hydrolysis coupled to kinesin G-protein superfamily head motor movement?

Answer 19

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.

Question 20

What is the powerstroke of kinesin motor?

Answer 20

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)

Question 21

What stimulates the release of ADP from kinesin leading head domain? (allowing the binding of ATP for neck-linker docking powerstroke)

Answer 21

Binding of the microtubule polymer stimulates ADP release from the kinesin.

Question 22

What 2 things determine directionality of motor movement?

Answer 22

1) Directionality of protofilament binding
2) directionality of neck-linker powerstroke

Question 23

Cytoplasmic dynein structure?

Answer 23

(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)

Question 24

Single cytoplasmic Dynein motor structure?

Answer 24

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)

Question 25

Difference between paths of dynein and kinesin?

Answer 25

Dynein towards MT minus end (kinesin to plus). 16nm hand-over-hand steps like kinesin, but more variable, with some big (>30nm) and some backward steps!

**Dynein able to take side steps (off-axis) **onto other MT protofilaments! (may be important to avoid collisions with kinesin)

Question 26

Kinesin, dynein and myosin all have high duty ratios, what does this mean?

Answer 26

Duty ratio is the fraction of time that a motor is attached to its filament

Question 27

3 Types of myosin that we have discussed?

Answer 27

Type II myosin: muscle myosin

Type V myosin: membrane trafficking, moves to plus end of actin

Type VI myosin: weird non-muscle myosin: unusual myosin moves wrong way down actin (towards minus)

Question 28

Type V myosin structure?

Answer 28

C-terminal light chain 8 membrane cargo binding region.

Heavy chain dimers join by coiled coil tail that leads neck and head groups at n-terminus.

Neck lever arms of Myosin V have 6 light-chain binding IQ domains.

Myosin head groups are in G-protein superfamily, bind ATP to release from actin.

Question 29

How does Myosin V move?

Answer 29

Towards plus end of actin with huge 72nm hand-over-hand steps. (cargo step size 36nm)

ATPase cycle changes conformation of light chain converter domain which is amplified by long light-chain binding lever arm!

(ATP binding releases myosin head from actin.)

Question 30

Differences between Myosin VI and Myosin V?

Answer 30

Myosin VI is weird, it moves towards the minus end of actin. (unlike all other myosins that move toward plus end)

It moves in reverse direction because it has special insert domains by its head groups that reverse direction of power stroke.

Although VI only has 2 light chain binding IQ domains (to V’s 6) it has a lever arm extending proximal tail. (allowing it to move the same 36nm step size)

Question 31

What is an axoneme?

Answer 31

Cytoskeletal structure of cilia or flagella, made up of microtubules.

(rings of 9, +0/2 cilia/flagella)

Question 32

How do cilia or flagella bend?

Answer 32

By dyneins moving A-tubule MT “cargo” along B-tubule track. (within axoneme)

Question 33

How do peri-trichous (flagella covered) bacteria swim?

Answer 33

Flagella change conformation and either bundle together for forward swimming. Repel each other to spread out to block swimming.

Question 34

Basic structure of flagella?

Answer 34

Filament inserts into hook, which inserts into rotating basal body (made of 2/4 rings of integral proteins) via a rod.

Filament can therefore rotate 360deg powered by PMF across membrane!

(conformational switching between more Left or more Right handed coil changes direction of swimming)