http://192.168.1.40:8080/xmlui/handle/123456789/1124 2026-05-31T10:20:00Z 2026-05-31T10:20:00Z Datta, A. B. Roy, S. Parrack, Pradeep http://192.168.1.40:8080/xmlui/handle/123456789/2324 2013-03-22T05:37:39Z 2005-01-14T00:00:00Z

Role of C-terminal residues in oligomerization and stability of λ CII: Implications for lysis-lysogeny decision of the phage Datta, A. B.; Roy, S.; Parrack, Pradeep A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN(6)TTGC at each. The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions. Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different. The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed. DOI: 10.1016/j.jmb.2004.09.098

2005-01-14T00:00:00Z Chattopadhyay, R. Parrack, Pradeep http://192.168.1.40:8080/xmlui/handle/123456789/2217 2013-03-14T07:41:51Z 2006-03-01T00:00:00Z

Cyclic AMP-dependent functional forms of cyclic AMP receptor protein from Vibrio cholerae Chattopadhyay, R.; Parrack, Pradeep The cyclic AMP receptor protein (CRP) from Escherichia coli, involved in the transcriptional regulation of a number of genes and operons, works by binding to specific sites upstream of promoters. CRP also binds cyclic AMP (cAMP), and this binding, which causes conformational changes in CRP, is mandatory for its activity. A cAMP-dependent variation in the conformation as well as biological activity of E coli CRP has been reported, with the cAMP-CRP complex formed at high cAMP concentrations resembling the uncomplexed apoprotein CRP. CRP from Vibrio cholerae, which plays an important role in the regulation of virulence gene expression, has a 95% sequence identity with the E coli protein. We have purified and characterized V. cholerae CRP and studied its transcription activation properties as a function of increasing cAMP concentrations. A biphasic dependence on cAMP levels was observed, similar to that found for E.coli CRP. The implications of these results on regulation of cAMP-CRP dependent promoters in V cholerae has been discussed. DOI: 10.1016/j.abb.2006.01.001

2006-03-01T00:00:00Z HaIder, Sabyasachi Datta, Ajit Bikram Parrack, Pradeep http://192.168.1.40:8080/xmlui/handle/123456789/2076 2013-03-05T05:22:59Z 2007-11-01T00:00:00Z

Probing the antiprotease activity of lambda CIII, an inhibitor of the Escherichia coli metalloprotease HflB (FtsH) HaIder, Sabyasachi; Datta, Ajit Bikram; Parrack, Pradeep The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor lambda CII, thereby helping the phage to lysogenize the host bacterium. lambda CIII, a small (54-residue) protein of unknown structure, also protects sigma(32), another specific substrate of HflB. In order to understand the details of the inhibitory mechanism of CIII, we cloned and expressed the protein with an N-terminal six-histidine tag. We also synthesized and studied a 28-amino-acid peptide, CHIC, encompassing the central 14 to 41 residues of CIII that exhibited antiproteolytic activity. Our studies show that CIII exists as a dimer under native conditions, aided by an intersubunit disulfide bond, which is dispensable for dimerization. Unlike CIII, CHIC resists digestion by HflB. While CIII binds to HflB, it does not bind to CII On the basis of these results, we discuss various mechanisms for the antiproteolytic activity of CIII. DOI: 10.1128/JB.00820-07

2007-11-01T00:00:00Z Jain, Nikhil Dhimole, Neha Khan, Abu Rafay De, Debojyoti Tomar, Sushil Kumar Sajish, Mathew Dutta, Dipak Parrack, Pradeep Prakash, Balaji http://192.168.1.40:8080/xmlui/handle/123456789/1918 2013-02-21T07:45:39Z 2009-02-06T00:00:00Z

E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides Jain, Nikhil; Dhimole, Neha; Khan, Abu Rafay; De, Debojyoti; Tomar, Sushil Kumar; Sajish, Mathew; Dutta, Dipak; Parrack, Pradeep; Prakash, Balaji HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA - the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is Stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding. DOI: 10.1016/j.bbrc.2008.12.072

2009-02-06T00:00:00Z