Recently a mechanism for determining the rate at which translocation occurs has been demonstrated and depends upon the enzyme's ability to displace an oligonucleotide that forms a triple helix (Firman & Szczelkun, 2000). The enzyme is able to translocate 400bp/sec in both directions and the process is extremely processive. This bulk biochemistry has been confirmed by the first single-molecule studies of these enzymes using a Magnetic Tweezer Set-up (Seidel et al., 2004 - see picture opposite), which has described in greater detail the mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration. They were able to show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, they also found that the enzyme tracks along the helical pitch of the DNA molecule.
The
translocation process by an EcoR124I enzyme containing a
single motor (HsdR) subunit shows frequent re-setting with
release of the magnetic bead. In fact, it is this
resetting process that is responsible for the lower
translocation processivity of the R1-complex and
Seidel
et al (2004) were able to demonstrate this resetting
process involves dissociation of the HsdR subunit.
More recent studies have shown that the MTase remains bound
to the target recognition sequence for >1800sec, that
re-binding of HsdR is required for translocation, but that
initial loop formation (see above) is the rate-limiting step.
The stalling events allow the motor to reset the direction of translocation and appear to involve release of the DNA. In fact by arranging the substrate such that the distance to the bead is less than the distance to the surface to which the DNA is bound, these events can be easily distinguished and the 85nm events shown opposite are translocation to the bead followed by stalling and release of the DNA (Seidel et al 2004).
The
figure below shows the results of an experiment in which the
R1-complex
is translocating on negatively supercoiled DNA. The DNA is supercoiled in the
presence of enzyme and ATP.
After random time translocation starts and the
enzyme induces positive supercoils. Therefore, plectonemes of the negative
supercoiled DNA are released resulting in an increase of the DNA end-to-end
distance. At maximum position where all supercoils are released, the actual
translocated distance is 67 ± 28 nm
as indicated in the Figure. This corresponds to 10 ± 4 bp traveled distance
per induced supercoil, consistent with the enzyme tracking along the helical
pitch. Inset: DNA end-to-end distance versus magnet rotations for the same
molecule, but in the absence of enzyme. The length decrease results from the
formation of plectonemes.
The R2-complex was found to show what appears at first sight to be a highly complex pattern of bead movement. However, careful analysis of the individual traces shows that there are two separate events, which are produced by independent translocation of the two HsdR motor subunits. Each motor is able to translocate at approx 500bp sec-1, but can also 'stall' at any time. The motors can the reiniate translocation or release the DNA and then rebind and reinitiate translocation. This results in the complex pattern of translocation shown above (Seidel et al 2004). In contrast, R1-complex is less processive, but produces a more simple picture of translocation.
Last modified on
23 December 2011
© Dr Keith Firman
Author Dr Keith Firman.