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.
 PREVIOUS
PAGE
 BACK TO
POSTER PAGE
| 
