Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity

<p>Abstract</p> <p>Background</p> <p>Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor.</p> <p>Results</p> <p>Using highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to <it>O</it>₆₄and <it>O</it>_{<it>L </it>}types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C.</p> <p>Conclusion</p> <p>Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.</p

Interaction of Chandipura Virus N and P Proteins: Identification of Two Mutually Exclusive Domains of N Involved in Interaction with P

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N0) form (N0-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0

Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1

The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the 131st proline residue of the wildtype repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative Nterminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor

Elucidation of functional domains of Chandipura virus nucleocapsid protein involved in oligomerization and RNA binding: implication in viral genome encapsidation

Chandipura virus, a member of the vesiculovirus genera, has been recently recognized as an emerging human pathogen. Previously, we have shown that Chandipura virus Nucleocapsid protein N is capable of binding to both specific viral leader RNA as well as non-viral RNA sequences, albeit in distinct monomeric and oligomeric states, respectively. Here, we distinguish the regions of N involved in oligomerization and RNA binding using a panel of deletion mutants. We demonstrate that deletion in the N-terminal arm completely abrogates self-association of N protein. Monomer N specifically recognizes viral leader RNA using its C-terminal 102 residues, while oligomerization generates an additional RNA binding surface involving the N-terminal 320 amino acids of N overlapping with a protease resistant core that is capable of forming nucleocapsid like structure and also binding heterogeneous RNA sequences. Finally, we propose a model to explain the mechanism of genome encapsidation of this important human pathogen

A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42° C. While 40-95% operator-binding activity was shown to be retained at 35 to 42° C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38° C, although the latter showed only 10% less binding compared to that of the former at 32° C. The CIts391 showed almost no binding at 42° C. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42° C. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32° C. Interestingly, the repressoroperator complexes preformed at 0oC have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32° C after preincubation at 42 to 52° C. All these data suggest that the 131st proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end

Schematic representation of the domains of CHPV N involved in interaction with P and their functional importance.

<p>Binding of P to nascent N masks the N-N self association region of CHPV N (N⁰-P complex formation) and also blocks non-specific RNA binding (upper panel). N⁰ is capable of specifically recognizing the viral leader sequence and the C-terminal 102 amino acids are essential for this recognition. Therefore, in the monomeric form, N specifically recognizes the leader RNA, to form the nucleation complex. Subsequently, the process of N-N self-association begins and P is released. Upon oligomerization, a new RNA binding cavity is formed utilizing the N-terminal arm (1–47 aa) and the central region of N (lower panel). Thus, the phase of non-specific encapsidation begins. Once nucleocapsids have formed, P can again interact with N, this time with the C-terminal region of oligomeric N, to usher the viral polymerase (L) onto its template.</p

Oligonucleotides used for the construction of the GFP fused truncated N proteins.

<p>Oligonucleotides used for the construction of the GFP fused truncated N proteins.</p

N protein utilizes two separate domains for interacting with P in its monomeric and oligomeric forms.

<p>N-terminally His-tagged P protein (His-P) was allowed to interact with either wild-type N or different N mutants in 100 mM NaCl TET buffer containing 10 mM Imidazole for 30 minutes at 4°C. Reaction mixtures were applied to Ni-NTA column and elution profile assayed by silver staining. L- loading; F- flow through; W- 10 mM Imidazole wash; E- 250 mM Imidazole elution. (A) In the absence of 1% DOC treatment. Bovine Serum Albumin (BSA) was used as negative control. (B) Wild-type N or N mutants were pre-incubated with 1% DOC for 30 minutes, followed by dialysis in presence of His-P, before applying to Ni-NTA column. Samples were resolved in 12% SDS-PAGE and visualised by Coomasie brilliant blue staining.</p