Rho translocation at the single-molecule level

Rho is a ring-ATPase responsible for the general modulation of gene expression in Escherichia coli. We investigate the mechanism by which Rho translocates single-stranded RNA at the single-molecule level.

HFSP Career Development Award holder Marcelo Nollmann and colleagues
authored on Fri, 22 May 2015

Rho is a hexameric ring motor that uses the energy of ATP hydrolysis to translocate single-stranded RNA to terminate transcription. Transcription termination is a process whereby nascent mRNA is released from RNA polymerase to engage the subsequent steps of protein synthesis. In bacteria, Rho-dependent termination represents ~50% of termination events. In addition, Rho is an essential, general modulator of gene expression in E. coli (and, probably, in many other bacteria) where it also appears to control the expression of non-coding RNAs (sRNAs and antisense RNAs), suggesting a possible role in the bacterial response to environmental stresses or in bacterial virulence. The molecular mechanism of Rho translocation on single-stranded RNA is key to understanding how Rho manages to dissociate the transcript from the RNA polymerase. 

Figure: (left) A magnetic trap consists of a magnetic bead (green sphere) tethered to a glass surface by one tether molecule (black helix and blue line). Magnets above the sample (red triangles) can be translated to exert tension on the DNA molecule. RNA looping by Rho (orange sphere) leads to the shortening of tether extension, monitored by the change in height of the magnetic particle detected through an inverted objective (white rhombus). (Right) Representative trace of the change in RNA extension due to single-molecule Rho activity on a tether molecule containing a Rut site and in presence of ATP. Forward and backward velocities as well as pause length and processivity for each event can be obtained in the extension versus time curve.

Rho binds/assembles onto a specific site of the nascent RNA transcript called rut before activating its translocation activity. Using a combination of single-molecule nano-manipulation and single-molecule fluorescence assays allowed us, for the time, to monitor in real-time the translocation activity of Rho at the single-molecule level. This observation is only consistent with a tethered-tracking translocation model, in which Rho remains anchored to the rut site while extruding a loop of single-stranded RNA.  

The use of single molecule methods allowed us to directly observe the translocation phase of the reaction, and thereby to measure velocity and processivity of movement. The velocity of Rho translocation supports a ‘kinetic coupling’ between Rho and RNA polymerase during Rho-dependent termination. The processivity is larger than the average distance between rut sites and termination sites, further strengthening the tethered-tracking model. Finally, our results suggest that forces applied on the nascent RNA transcript by cellular substructures could have important implications for the regulation of transcription and its coupling.

References

Direct observation of the translocation mechanism of transcription termination factor Rho. Gocheva V, Le Gall A, Boudvillain M, Margeat* E, Nollmann* M. Nucleic Acids Research, 2015 Feb 27; 43(4):2367-77.

DNA segregation and remodelling group

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