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Transcription


Current Research Interests

Laboratory of transcription is engaged in understanding the mechanism of transcription termination and antitermination in prokaryotes.  A wide range of techniques from biophysics (spectroscopy, thermodynamics, kinetics etc.), biochemistry (protein purification, chemical and enzymatic footprinting of protein and nucleic acids, crosslinking etc.), molecular biology (recombinant DNA techniques, site-directed mutagenesis) and bacterial genetics are used in the laboratory to solve this challenging problem. Following projects are in progress.

  1. Mechanism of transcription termination by transcription termination factor Rho.
  2. Mechanism of transcription antitermination by the antiterminator protein, N.
  3. Mechanism of inhibition of Rho dependent termination by Psu.

Extramural Funding

  1. Wellcome Trust Senior Research Fellowship (2003-2008).
  2. DBT grant (2007-2010).
  3. Grant from DBT COE on "Microbial Physiology" (2008-2013).
  4. DST Swarnajayanti Fellowship (2008-2013; to be activated).

Research Highlights


A) Transcription termination factor Rho prefers catalytically active elongation complexes for releasing RNA (JBC, 2008).

RNA polymerase (RNAP) 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 e 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.


B) The Site of Action of the Antiterminator Protein N from the Lambdoid Phage H-19B (JBC, 2007).

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 alambdoid 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, b (G1045D), and b (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 RNAhairpin-b 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.


C) Transcription Termination Defective Mutants of Rho: Role of Different Functions of Rho in Releasing RNA from the Elongation Complex (JMB, 2007).

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 structurefunction 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.

Projects in progress

  1. Rho-NusG interaction.
  2. Rho-Psu interaction.
  3. Mechanism of Antitermination of Rho-dependent termination by N protein.
  4. Characterization of Rho and NusG from M.Tb.

Projects to be initiated:

  1. Fast kinetics approaches to follow termination processes using stopped flow/quench flow methods.
  2. In vivo role of transcription termination factor Rho using genomics approaches.
 
Dr. Ranjan Sen
Transcription Group
Publications

CONTACT INFORMATION
Email :
 rsen@cdfd.org.in
Phone : 
+91-40-27151344 extn: 1403
Fax     :
+91-40-27151344
Last updated on : 22nd May, 2008.