Molecular Machines

Question 1

What are the features of the palm, fingers & thumb?

What are the 5 cyclisation steps?

What is the rate limiting step?

Where can the cycle be re-entered? 2 ways

Answer 1

Active site, close on nucleotide for bonding, structural

1. E + p/t -> E:p/t (polymerase binds to primer template)
2. E:p/t : dNTP binds dNTP
3. E:p/t:dNTP -> E’:p/t:dNTP conformational change
4. E’:p+1/tPPi elongation of primer by 1 base
5. E:p+1/t + PPi biphosphate forms & E’ to E

E -> E’

E:p/t dissociates from dNTP so from 2
E:p/t:dNTP stays bound to dNTP so from 3

Question 2

What did comparison of the E & E:p/t states show? 2

E:p/t and E:p/t:dNTP?

E:p/t:dNTP and E’p/t:dNTP?

How was this last step achieved?

Answer 2

1. Where polymerase binds to p/t
2. DNA constrained in 1 motion - which also prevents polymerase falling off

dNTP binds to o-helix which is 20A from catalytic region

O-helix moves from open state to closed state such that dNTP is delivered to the palm/active site

Removed 5’OH from primer such that the dNTP was stuck but no phosphodiester bond formed & crystallised this

Question 3

What else is involved in the reaction mechanism?

Answer 3

2 Mg2+ - in coordination with Asp and OH in tetrahedral arrangement for reaction

Question 4

What are Type I & P pili involved in?

What are the pilus made up of?

How is the Pap operon organised?

How does a chaperone bind to a pilus subunit?

What fold does it complete?

How is pilus assembly terminated?

What is the process called through which the pilus assembles?

Answer 4

Attaching E. coli to human kidneys & bladders

Rod & tip fibrilum

Subunits of the pilus formed in defined order - fibrilum, rod subunits, rod terminator, usher, chaperones

Pilus subunit lacks beta strand G - so chaperone binds & subunits inserts its N-terminal extension into subunit groove to stabilise

Ig fold

Termination subunit inserts N-terminal extension in but doesn’t produce Ig fold - so other subunits cannot insert into it

Donor strand exchange

Question 5

What are the 3 features of the usher?

What are the 3 domains of the usher?

What are the functions of these domains?

Why is the type 1 pilus easier to image?

Answer 5

1. Outer membrane assembly platform
2. Outer membrane secretion pore
3. Large protein

N-terminal, translocation domain 500AA, C-terminal

N = binds subunit-chaperone complex (recruits)
T = forms 24 strand peripore to control contents in/out bacteria
C = unknown

Only has 3 rod subunits instead of 5 - easier & less complex

Question 6

Through co-expression of Usher, chaperones & tip subunits, what did they discover about the C-terminal domain? 2

What does the NTD do?

What are the 5 steps to pilus assembly/donor strand exchange?

Answer 6

1. 2 c-terminal domains
2. Chaperone-subunit complex binds to CTD1 & 2

Recruits chaperone-subunit complex - passes to CTD so available for next chaperone-subunit complex

1. Chaperone-subunit complex binds to the NTD of the Usher & subunit N-terminal extension fits into the groove of the green subunit
2. Chaperone unbinds from the CTD
3. Complex moves to CTD (chaperone unbinds from CTD) which frees the NTD of the Usher to bind the next chaperone-subunit complex
4. Red subunit positions its N-terminal extension into the groove of the orange subunit - recruited by the NTD of the Usher 5. Chaperone unbinds to CTD site is free so complex moves to CTD so NTD of Usher is free