The biological macromolecular world is homochiral and effective enforcement and perpetuation of this homochirality is essential for cell survival. In this study, we present the mechanistic basis of a configuration-specific enzyme that selectively removes D-amino acids erroneously coupled to tRNAs. The crystal structure of dimeric D-aminoacyl-tRNA deacylase (DTD) from Plasmodium falciparum in complex with a substrate-mimicking analog shows how it uses an invariant ‘cross-subunit’ Gly-cisPro dipeptide to capture the chiral centre of incoming D-aminoacyl-tRNA. While no protein residues are directly involved in catalysis, the unique side chain-independent mode of substrate recognition provides a clear explanation for DTD’s ability to act on multiple D-amino acids. The strict chiral specificity elegantly explains how the enriched cellular pool of L-aminoacyl-tRNAs escapes this proofreading step. The study thus provides insights into a fundamental enantioselection process and elucidates a chiral enforcement mechanism with a crucial role in preventing D-amino acid infiltration during the evolution of translational apparatus.

The transcription termination factor Rho of Escherichia coli is a RNA binding protein which can translocate along the RNA and unwind the RNA:DNA hybrid using the RNA-dependent ATPase activity. In order to investigate the involvement of each of these functions in releasing RNA from the elongation complex, we have isolated different termination defective mutants of Rho by random mutagenesis, characterized them for their different functions and established the structure-function correlations from the available structural data of Rho. These mutations are located within the two domains; the N-terminal RNA binding domain (G51V, G53V, and Y80C) and in the C-terminal ATP binding domain (Y274D, P279S, P279L, G324D, N340S, I382N) including the two important structural elements, the Q-loop (P279S, P279L) and R-loop (G324D). Termination defects of the mutants in primary RNA binding domain and Q-loop could not be restored under any conditions that we tested and these were also defective for most of the other functions of Rho. The termination defects of the mutants (Y274D, G324D and N340S), which were mainly defective for secondary RNA binding and likely defective for translocase activity, could be restored under relaxed in vitro conditions. We also show that a mutation in a primary RNA binding domain (Y80C) can cause a defect in ATP binding and induce distinct conformational changes in the distal C-terminal domain, and these allosteric effects are not predictable from the crystal structure. We conclude that the interactions in the primary RNA binding domain and in the Q-loop are mandatory for RNA release to occur and propose that the interactions in the primary RNA binding modulate most of the other functions of Rho allosterically. The rate of ATP hydrolysis regulates the processivity of translocation along the RNA and is directly correlated with the efficiency of RNA release. NusG improves the speed of RNA release and is not involved in any other step.

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.

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.