Dr Abhishek Mazumder

Abstract:

RNA polymerase (RNAP) contains a mobile structural module, the ‘clamp,’ that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including promoter melting, transcription elongation complex stability, transcription pausing, and transcription termination. Using single-molecule FRET on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme populates three distinct conformational states and interconvert between these states on the 0.1–1 s time-scale. Similar studies confirm that the RNAP clamp is closed in open complex (RPO) and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, the clamp does not exhibit dynamic behaviour. We also show that, the stringent-response alarmone ppGpp, which reprograms transcription during amino acid starvation stress, selectively stabilizes the partly-closed-clamp state and prevents clamp opening; these results raise the possibility that ppGpp controls promoter opening by modulating clamp dynamics.

Abstract:

A peptide-based cell permeable synthetic transcription factor is reported, which binds to its target site with high affinity and specificity. When linked to a HAT-binding peptide, it causes significant upregulation of gene expression in a mammalian cell. Such molecules may be developed for selectively activating repressed genes in mammalian cells.

Abstract:

Most genes are regulated by multiple transcription factors, often assembling into multi-protein complexes in the gene regulatory region. Understanding of the molecular origin of specificity of gene regulatory complex formation in the context of the whole genome is currently inadequate. A phage transcription factor λ-CI forms repressive multi-protein complexes by binding to multiple binding sites in the genome to regulate the lifecycle of the phage. The protein-protein interaction between two DNA-bound λ-CI molecules is stronger when they are bound to the correct pair of binding sites, suggesting allosteric transmission of recognition of correct DNA sequences to the protein-protein interaction interface. Exploration of conformation and dynamics by time-resolved fluorescence anisotropy decay and molecular dynamics suggests a change in protein dynamics to be a crucial factor in mediating allostery. A lattice-based model suggests that DNA-sequence induced allosteric effects could be crucial underlying factors in differentially stabilizing the correct site-specific gene regulatory complexes. We conclude that transcription factors have evolved multiple mechanisms to augment the specificity of DNA-protein interactions in order to achieve an extraordinarily high degree of spatial and temporal specificities of gene regulatory complexes, and DNA-sequence induced allostery plays an important role in the formation of sequence-specific gene regulatory complexes.

Abstract:

A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.

Abstract:

An overwhelming number of structural and functional studies on specific protein-DNA complexes reveal the existence of water molecules at the interaction interface. What role does the interfacial water molecules play in determining the specificity of association is thus a critical question. Herein, we have explored the dynamical role of minor groove water molecules and DNA side chain flexibility in lambda repressor-operator DNA interaction using well-characterized DNA minor groove binder dye, Hoechst 33258. The most striking finding of our studies reveals that the solvation time scale corresponding to the minor groove water molecules (∼50 ps) and DNA side chain flexibility (∼10 ns) remain unaltered even in protein-DNA complex in comparison to unbound operator DNA. The temperature dependent study further reveals the slower exchange of minor grove water molecules with bulk water in DNA-protein complex in comparison to the unbound DNA. Detailed structural studies including circular dichroism (CD) and Förster resonance energy transfer (FRET) have also been performed to elucidate the interaction between protein and DNA.