Cooperative Assembly of Terminase and Integration Host Factor at the Packaging Initiation Site of Bacteriophage Lambda
Sanyal, Saurarshi Jyoti
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Packaging of viral genomes into procapsids by terminase enzymes is conserved in many DNA viruses. Terminases bind to linear concatemers of replicated viral genomes and concomitantly excise (mature) and package a single genome per procapsid. In this thesis, I interrogate the role of E. coli integration host factor (IHF) in mediating the site-specific assembly of bacteriophage lambda terminase at its cognate DNA site, cos, which serves as the packaging initiation site. IHF binds to an I-element within cos and introduces a strong bend in the duplex. It was previously demonstrated that the small terminase subunit could stabilize an IHF-induced bend at cos. I hypothesized that terminase holoenzyme and IHF cooperatively assemble at cos and wrap the duplex into a compact nucleoprotein complex. Rigorous analysis of this cooperative assembly is complex due to the multiple terminase and IHF binding elements within cos. Therefore, I dissected the cos site into individual specific and nonspecific IHF binding sequences, and analyzed the relevant protein affinities for these subsites as well as for (1) the full-length cos site and (2) a random nonspecific (NS) sequence of equivalent length. Analytical ultracentrifugation and electrophoretic mobility shift studies show that IHF and terminase only modestly discriminate between cos and NS-DNA substrates; however, the two proteins cooperatively bind to cos-DNA. The data suggest that IHF confers site-specificity of binding to terminase. Also evident is significant nonspecific DNA binding concurrent with specific interactions, even on specific DNA substrates. IHF likely facilitates the high-affinity cooperative assembly of a relevant nucleoprotein complex at the cos site despite significant nonspecific binding of both proteins to DNA, with the functional significance of nonspecific DNA binding being the enhancement of protein-DNA interactions. Furthermore, sedimentation equilibrium studies demonstrate that while terminase assembles in the absence of IHF as a dimer on a 274 bp DNA substrate inclusive of the entire cos site, in the presence of IHF a nucleoprotein complex of mass consistent with five terminase protomers and two IHF molecules results. This finding further implicates IHF in the cooperative assembly of a specific ternary IHF-DNA-terminase complex at the packaging initiation site of bacteriophage lambda. A terminase packaging enzyme that both (1) site-specifically matures DNA and (2) packages DNA in a sequence-independent manner must be capable of both specific and nonspecific DNA binding. This work furthers the understanding of (1) one of the factors (IHF) involved in the site-specific assembly of a nucleoprotein complex required for the initiation of viral packaging, and (2) the nature of the specific nucleoprotein complex assembled at the cos site prior to DNA maturation and packaging.
- Medicinal chemistry