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.

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© University of Portsmouth
Author Dr Keith Firman,
Page last updated September 28, 2008