Ranjan Sen

Interaction Surface of the Transcription Terminator Rho Required to Form a Complex with the C-Terminal Domain of the Antiterminator NusG

Rho-dependent transcription termination in bacteria requires an interaction between the terminator Rho and the antiterminator NusG. The interaction surface of the Rho-NusG complex is unknown. Here we provide direct evidence that the β-sheet bundle of the C-terminal domain of NusG (NusG-CTD) has the binding determinants for Rho, proving the hypothesis described earlier [Mooney, R. A., Schweimer, K., Rosch, P., Gottesman, M., & Landick, R., (2009). Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. J. Mol. Biol., 391, 341-358.]. Disulfide bridges can be engineered from NusG-CTD with the surface-exposed amino acids 217 and 224 of Rho, which belong to its P-loop ATPase domain. Mutational analyses of this region of Rho revealed that a hydrophobic pocket, located behind these amino acids of Rho, is the docking site for NusG-CTD. The proximity of this region of Rho to NusG-CTD in the Rho-NusG complex was also confirmed by an efficient fluorescence resonance energy transfer between residue K224 of Rho and residue A168 of NusG-CTD. The identification of the Rho-NusG interaction surface will be useful not only in understanding the role of NusG in the termination process but also in explaining the molecular basis of the involvement of NusG-CTD in recruiting Rho and the ribosome to the same transcription machinery.

Transcription Termination Factor Rho Prefers Catalytically Active Elongation Complexes for Releasing RNA

RNA polymerase pauses at different DNA sequences during transcription elongation, and this pausing is associated with distinct conformational state(s) of the elongation complex (EC). Transcription termination by the termination factor Rho, an RNA-dependent molecular motor, requires pausing of the EC in the termination zone of Rho-dependent terminators. We hypothesized that the conformational state(s) of the EC associated with this pausing would influence the action of Rho. Analyses of the pausing behavior of the EC at the termination points of two well known Rho-dependent terminators revealed that Rho prefers actively transcribing complexes for termination. RNA release kinetics from stalled ECs showed that the rate of RNA release by Rho was reduced if the EC was irreversibly backtracked, if its RNA exit channel was modified by an RNA hairpin, or the bridge helix/trigger loop movement in its active site was perturbed. These defects were overcome significantly by enhancing the rate of ATP hydrolysis either by increasing the concentration of ATP or by using a Rho mutant with higher ATPase activity. We propose that the force generated from ATP hydrolysis of Rho is the key factor in dislodging the EC through its molecular motor action, and this process is facilitated when the EC is in a catalytically competent state, undergoing rapid “Brownian ratchet” motion.

Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.

Psu, a coat protein from bacteriophage P4, has been shown to inhibit Rho-dependent transcription termination in vivo. Co-overexpression of Psu and Rho led to the loss of viability of the cells, which is the consequence of the anti-Rho activity of the protein. The antitermination property of Psu is abolished either by the deletion of 10 or 20 amino acids from its C terminus or by a mutation, Y80C, in Rho. All these experiments indicated probable interactions between Rho and Psu. Purified Psu protein is α-helical in nature and appeared to be a dimer. Co-purification of Rho and wild-type Psu on an affinity matrix and co-elution of both of them in Superose-6 gel filtration suggests a direct association of these proteins, whereas a C terminus 10-amino acid deletion derivative of Psu failed to be pulled down in this assay. This indicates that the loss of the function of these mutants is correlated with their inability to interact with each other. In vitro termination assays revealed that Psu can inhibit Rho-dependent termination specifically in a concentration-dependent manner. The presence of Psu affected the affinity of ATP and reduced the rate of ATPase activity of Rho but did not affect either primary or secondary RNA binding activities. In the presence of Psu, Rho was also observed to release RNA very slowly from a stalled elongation complex. We propose that Psu inhibits Rho-dependent termination by slowing down the translocation of Rho along the RNA because of its slow ATPase activity.

Interaction with the nascent RNA is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro.

In the conventional model of the Rho-dependent transcription termination, the terminator Rho binds to the rut (Rho utilization) site and translocates along the nascent RNA prior to making possible interactions with the elongating RNA polymerase (RNAP). Even though the interaction between Rho and isolated RNAs was studied in great detail, the same has never been shown with the nascent RNA emerging from the transcription elongation complex (EC). Direct demonstration and requirement of the Rho-nascent RNA binding become even more important because of the recently proposed alternative model where Rho loads onto the RNAP prior to the formation of the nascent RNA. Here, we have measured the direct association of Rho in vitro with the free RNAP, RNAP-promoter binary complex and stalled ECs with varied length of RNA. We observed the association of Rho only with the ECs having the rut-site-containing long nascent RNA. This association was significantly reduced when either a Rho mutant, Y80C, defective for RNA binding or an antisense oligo to the rut site was used or when the rut site was eliminated by RNase digestion or replacement with a random RNA sequence. The presence of EC-bound NusG, the binding partner of Rho, did not facilitate this association. RNase footprinting of the Rho-EC complex revealed a clear Rho-mediated protection of the rut sites on the nascent RNA. We concluded that the nascent RNA loading of Rho and its interaction with the rut site are mandatory and prerequisites for its recruitment to the EC under in vitro experimental conditions.

A bacterial transcription terminator with inefficient molecular motor action but with a robust transcription termination function.

Molecular motors such as helicases/translocases are capable of translocating along the single-stranded nucleic acids and unwinding DNA or RNA duplex substrates using the energy derived from their ATPase activity. The bacterial transcription terminator, Rho, is a hexameric helicase and releases RNA from the transcription elongation complexes by an unknown mechanism. It has been proposed, but not directly demonstrated, that kinetic energy obtained from its molecular motor action (helicase/translocase activities) is instrumental in dissociating the transcription elongation complex. Here we report a hexameric Rho analogue (Rv1297, M. tb. Rho) from Mycobacterium tuberculosis having poor RNA-dependent ATP hydrolysis and inefficient DNA-RNA unwinding activities. However, compared to Escherichia coli Rho, it exhibited very robust and earlier transcription termination from the elongation complexes of E. coli RNA polymerase. Bicyclomycin, an inhibitor of ATPase as well as RNA release activities of E. coli Rho, inhibited the ATPase activity of M. tb. Rho with comparable efficiency but was not efficient in inhibiting its transcription termination function. Unlike E. coli Rho, M. tb. Rho was capable of releasing RNA in the presence of nonhydrolyzable analogues of ATP quite efficiently. Also, this termination function most likely does not require NusG, an RNA-release facilitator, as this Rho was incapable of binding to NusG either of M. tb. (Rv0639) or E. coli. These results strongly suggest that the ATPase activity of M. tb. Rho is uncoupled from its transcription termination function and this function may not be dependent on its helicase/translocase activity.

Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho

The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions.

Random Mutagenesis Using a Mutator Strain

Random mutagenesis is a useful technique to study the functions of different gene products. Propagation of the genes cloned in plasmids through a mutator strain, like Escherichia coli XL1-red, produces randomly mutagenized plasmid libraries. This method offers a very simple and economic way of introducing random point mutations throughout the gene with a fairly high mutation rate. The whole process involves transformation and propagation of a plasmid containing the desired gene into the XL-1 red strain, isolating the mutagenized plasmid library and transforming this library into a desired strain for screening the mutant phenotype.

Nus Factors of Escherichia coli.

The highly conserved Nus factors of bacteria were discovered as essential host proteins for the growth of temperate phage λ in Escherichia coli. Later, their essentiality and functions in transcription, translation, and, more recently, in DNA repair have been elucidated. Close involvement of these factors in various gene networks and circuits is also emerging from recent genomic studies. We have described a detailed overview of their biochemistry, structures, and various cellular functions, as well as their interactions with other macromolecules. Towards the end, we have envisaged different uncharted areas of studies with these factors, including their participation in pathogenicity.

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

The Site of Action of the Antiterminator Protein N from the Lambdoid Phage H-19B

Transcription antitermination by N proteins of lambdoid phages involves specific interactions of the C-terminal domain of N with the elongation complex (EC). The interacting surface of N on the EC is unknown, knowledge of which is essential to understand the mechanism of antitermination. Specific cleavage patterns were generated near the active site Mg2+of the RNA polymerase of an N-modified stalled EC using iron-(S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate conjugated to the only cysteine residue in the C-terminal domain of N from a lambdoid phage H-19B. Modification of EC by N also induced conformational changes around the same region as revealed from the limited trypsin digestion and in situ Fe-dithiothreitol cleavage pattern of the same EC. These data, together with the previously obtained H-19B N-specific mutations in RNA polymerase, β (G1045D), and β′ (P251S, P254L, G336S, and R270C) subunits, suggest that the active center cleft of the EC could be the site of action of this antiterminator. H-19B N induced altered interactions in this region of EC, prevented the backtracking of the stalled EC at the ops pause site and destabilized RNA hairpin-β subunit flap domain interactions at the his pause site. We propose that the physical proximity of the C-terminal domain of H-19B N to the active center cleft of the EC is required for the process of transcription antitermination and that it involves both stabilization of the weak RNA-DNA hybrid at a terminator and destabilization of the interactions of terminator hairpin in the RNA exit channel.