## 4 Rho-Dependent Termination  
Transcription termination factor Rho is an essential protein in _E. coli_ first identified for its role in transcription termination at Rho-dependent terminators [10], and is estimated to terminate \(\sim\)20% of _E. coli_ transcripts [11]. _rho_ is highly conserved and nearly ubiquitous in bacteria [12]. Rho is an RNA-dependent ATPase [13] with RNA:DNA helicase activity [14], and consists of a hexamer of six identical monomers arranged in an open circle [15]. Transcription of the single copy of _rho_ is regulated by Rho-dependent transcription termination at a Rho termination site located upstream of the structural gene [16]. Figure 1: Model of Intrinsic termination. Rho-dependent terminators are comprised of _rut_ sites (_r_ho _u_t_ilization) and release sites [17]. Rho binds with a high affinity to the _rut_ site RNA, is stimulated to hydrolyze ATP, and then translocates along the RNA in a 5' to 3' direction while maintaining an interaction with _rut_[18, 19], until it encounters TEC at a release site. Termination requires untranslated RNA of at least 85-97 nt [20]. RNA binds to two distinct sites in Rho, termed primary and secondary [21]. The primary site stably binds RNA in the absence of ATP. The secondary sites are stimulated to bind RNA transiently after the primary site is occupied. Secondary site binding stimulates ATP hydrolysis. Crystallography has identified the location of the primary site on the outer edge of the hexamer and the secondary site around the center hole [15, 22]. The Rho hexamer initially binds RNA in an open \"lockwasher\" conformation. After RNA is bound to the primary site, the transcript is threaded through the central hole contacting the secondary binding site and the hexamer closes (Figure 2, [23]). Single molecule experiments determined that Rho binds \(57\pm 2\) nucleotides of RNA in the absence of ATP and \(80\pm 2\) nucleotides upon ATP hydrolysis, consistent with 60 nt binding at the primary and 20 nt at the secondary site [19]. The transient interactions of RNA with the secondary site drive Rho translocation along the RNA until it encounters TEC at a release site. At this point, Rho releases RNAP from the template, presumably by unwinding the RNA-DNA hybrid. The precise mechanism of arrest and removal is unclear. [PERSON] _et al_. [24] propose that Rho causes a conformational change in RNAP leading to arrest of TEC and exposure of the transcription hybrid to Rho. In this model, Rho then unwinds the RNA-DNA hybrid, removing RNAP. The authors argue that since crosslinking data indicates that the active site does not move in relation to the template during transcription termination, forward translocation does not contribute to termination. [PERSON] and [PERSON] [25], however, found that Rho induces forward movement of TEC, and that mispairings in the DNA template immediately 5' to the arrested TEC decrease the efficiency of Rho termination. [PERSON] and [PERSON] propose that Rho induces termination by pushing RNAP ahead of the transcription hybrid. The precise mechanism of removal is thus still unclear, but the above studies suggest that forward pressure on RNAP from Rho causes a conformational change leading to arrest. Continued pressure then exposes the transcription hybrid to Rho either by a conformational change in RNAP or by removal without forward translocation. Termination may not entail specific interactions with RNAP, since _E. coli_ Rho factor will efficiently terminate transcription of _Saccharomyces cerevisiae_ RNA pol II [26]. Rho-dependent terminators, unlike intrinsic terminators, lack an easily identifiable motif. _rut_ sites are likewise not highly conserved. They consist of unstructured RNA that is C-rich and G-poor compared to the flanking sequences [20, 27, 28]. Cytidine residues most strongly activate the ATPase activity [13]. Release sites correlate with TEC pausing, but not all pause sites can function as efficient release sites [24, 29]. Efficient Rho-dependent termination _in vivo_ requires the NusG transcription factor [30].