Progress to date

Transfer of Techniques

This series of AFM images visualise, for the first time at a single molecule level, the assembly of an EcoR124I complex, allowing measurement of the size of the complex in its different states. This has allowed us to show that the EcoR124I system does not need to dimerise to allow translocation to take place, unlike the EcoKI system.

Figure 1 – AFM images were imaged on
poly-l-lysine coated mica. The 
protein complex was visualised bound to a 
758bp section of DNA with a single recognition
site 178bp from the end.
A) pCFD30 linearised with HindIII
B) MTase bound with the average size 85nm by
85nm.
C) R1complex bound with the average size of
bound R1 90nm by 85nm.
D) R2complex average size of bound R2 90nm
by 90nm
E) DNA translocation by EcoR124I produced
from a 1:1 molar ratio of 
HsdR(prrI):MTase  after 60 s. incubation with 
DNA and 5 mM ATP.

Conclusion

We have shown that the EcoR124I
system does not need to dimerise
for translocation to take place. The
system is able to bind and translocate in 
both R1 and R2-complexes. ATP is
necessary to see these translocation
events.  Translocation is preceded by
the formation of the initial bulge 
captured by ATPgS.

Future work

To progress the project I plan to construct a two site plasmid to investigate the 
effect of two complex’s binding in tandem which should help to give a better 
understanding of both translocation and possibly cleavage.

I will continue to optimise conditions for assembly on different surfaces as the project progresses towards the development of a biosensor.

Mutants will also be interesting to visualise, as hopefully they can provide even more structural information, which could help us to understand the phenotypes seen through other assays.

We intend to use AFM not only to study translocation and pre-translocation events, but also subunit assembly.  The aim is to develop a biosensor that is based upon interruption to self-assembly and detection of such events using a simple optical system.

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