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. Lab is funded
by NIH, Wellcome Trust, DBT and CDFD core funds.
Following projects are in
progress.
- Mechanism of transcription termination
by transcription termination factor Rho.
- Mechanism of transcription
antitermination by the antiterminator protein, N.
- Mechanism of inhibition of Rho dependent
termination by Psu.
Research Highlights |
1. Interaction surface of antiterminator protein N on
the elongation complex.
(funded by NIH,
grant# RO1 TW06185-01)
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 Mg+2of the RNAP of an N-modified stalled EC
using Fe-BABE 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-DTT cleavage
pattern of the same EC. These data, together with the previously
obtained H-19B N specific mutations in RNAP,
β(G1045D) and
β'(P251S, P254L,
G336S and R270C) subunits, broadly defines that the active center cleft
of the EC as the site of action of this antiterminator. H-19B N-induced
altered interactions in this region of EC, also prevented the
backtracking the stalled EC at 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 plays a key role
in the process of transcription antitermination by this antiterminator
and it is achieved by both stabilizing the weak RNA:DNA hybrid at a
terminator and by destabilizing the interactions of terminator hairpin
in the RNA exit channel [JMB (2005), 352, 28-43; JBC (2007), in
press].
2. Transcription termination defective mutants of Rho: role of
different functions of Rho in releasing RNA from the elongation complex
(funded by Wellcome trust senior research
fellowship).
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, 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 primary RNA binding domain (Y80C) can cause
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 [JMB (2007), 371, 855-872].
3.
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
alpha-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 [JBC (2006), 281,
26491-26500].
Projects in progress
- Transcription elongation complex
preference of Rho.
- Rho-NusG interaction.
- Rho-Psu interaction.
- Mechanism of Antitermination of
Rho-dependent termination by N protein.
- Characterization of Rho and NusG from
M.Tb.
Projects to be initiated:
- Fast kinetics approaches to follow
termination processes using stopped flow/quench flow methods.
- In vivo role of transcription
termination factor Rho using genomics approaches.
|
Dr. Ranjan Sen