## 4 Rho-Dependent Termination
NusG binds to Rho [31] and shifts termination to more 5' release sites [32, 33, 34]. Rho binds NusG weakly in solution (\(\mathrm{k_{D}=0.2\ \mu M}\); [31]) but NusG enhances Rho binding to RNAP _in vitro_[35]. ChIP-chip analysis reveals that Rho associates with the TEC throughout transcription, rather than only after synthesis of an untranslated _rut_ binding site [36]. [PERSON] _et al_. [37] instead maintain that a transcribed _rut_ element is required for Rho association with RNAP, and suggest that the ChIP-chip data does not reflect a relevant interaction between Rho and TEC. Single molecule studies show no evidence that Rho directly binds to RNAP [19], however, [PERSON] _et al_. [24] did detect direct binding to RNAP _in vitro_. The reason for the above discrepancy remains unclear. Rho-dependent termination in _E. coli_ occurs predominantly within the \"foreign\" DNA (e.g., cryptic prophages and transposons) that makes up \(\sim\)14% of the genome of _E. coli MG1655_[38]. Efficient transcription termination in _E. coli_ is essential to suppress expression of toxic genes in this horizontally-acquired DNA [38]. Although Rho activity can be significantly reduced by _musA_ or _musG_ mutations in a strain deleted of all horizontally transferred DNA, _rho_ cannot be deleted in this strain. [PERSON] and [PERSON] proposed that Rho is essential to prevent collisions between TEC and the replisome [39]. Rho termination within coding sequences may depend upon ribosome release by tmRNA, which can uncouple the lead ribosome from RNAP [40]. The RNA-DNA helicase activity of Rho can unwind extensive RNA-DNA hybrids. [PERSON] and [PERSON] [41] suggest that Rho prevents RNA-DNA hybrids (\"R-loops\") from forming between untranslated mRNA and the chromosome. In support of this hypothesis, [PERSON] _et al_. [42] found that _rho_ could be deleted in a _rac_- strain expressing the RNA-DNA helicase _uvsW_. This model assumes that both Rho and UvsW prevent or remove potentially lethal R-loops. Nevertheless, _rnhA_ deletion mutants are healthy despite extensive accumulation of R-loops. Nor are _rnhA_ mutants more dependent on _rho_ for survival than wild-type [43,44]. Perhaps Rho and UvsW share another essential activity, such as resolving conflicts between transcription and replication, or removing certain toxic R-loops sequestered from RNaseHI. Note that [PERSON] _et al_. [45], demonstrated that suppression of R-loop formation reduces transcription-replication clashes. [PERSON] _et al_. [46] found that inhibiting Rho or deleting _musG_ resulted in the accumulation of antisense transcripts. The antisense transcripts are untranslated, thus allowing Rho access to termination sites. Surprisingly, _nusA_ does not contribute to termination of the antisense transcripts, despite the similarities in transcription patterns in _nusG_, _nusA_ and cells treated with the Rho inhibitor bicyclomycin [38]. Whether or not this activity of Rho is important _in vivo_ is unclear. Figure 2: Structure and movement of Rho. The efficiency of Rho termination is dependent on the rate of transcription elongation [47]. Slow RNAP mutants have a decreased affinity for NTPs (e.g., _rpoB8_, 5-fold higher Km for ATP) and are more efficient at Rho-dependent termination. Conversely, fast mutants have an increased affinity for NTPs (e.g., _rpoB3595_) and are less efficiently terminated. Enhanced termination was observed _in vitro_ when the transcription rate was slowed by limiting NTP concentrations. This \"kinetic coupling\" model might explain the correlation between TEC pausing and Rho termination sites. The histone-like nucleoid-structuring protein H-NS contributes to Rho-dependent termination. _hns_ deletion mutations increase Rho dependency [43; 44], and H-NS is concentrated at the antisense transcription terminators [46]. This is distinct from the role of H-NS in silencing foreign genes by repressing promoters [48]. Horizontally-transferred DNA is AT rich compared to _E. coli_ sequences, which favors H-NS binding.