(i) Stereo-chemical role of pseudo-symmetric DNA operators in transcription regulation: The CI protein of the temperate bacteriophage Lambda is critical for stable maintenance of its prophage state in the Escherichia coli lysogen. CI binds to six binding sites (oR1, oR2, oR3, oL1, oL2 and oL3) on prophage DNA to both repress the cognate promoters, pL and pR, responsible for lytic growth, and to auto-regulate its own synthesis from an adjacent promoter, pRM. All six operators contain pseudo-symmetric sequences each with a consensus sequence (C) half and a non-consensus sequence (NC) half. Moreover, a CI homodimer bound to the asymmetric oR1 operator also assumes an asymmetric protein structure in which an extended subunit binds to the C-half and a condensed subunit binds to the NC-half. From modeling studies we proposed that the DNA-bound asymmetric CI dimer facilitates both co-operative binding to adjacent operators as well as establishing the contact between oR2-bound CI and pRM-bound RNA polymerase for transcription activation because of the stereo-chemically advantageous location of the extended CI subunit at oR2. Consistently, physico-chemical and biochemical experimental results showed that inversion of the oR2 operator from C-NC to NC-C orientation deranges both co-operative binding and pRM regulation. We showed that changing the orientation of oR1 operator from its normal state to an inverted state quantitatively decreases oR1-oR2 co-operative binding of CI, and changing the orientation of oR2 while substantially affects oR1-oR2 co-operativity, also significantly increases oR2-oR3 co-operativity and thus prevents pRM activation. These quantitative effects of operator inversions confirm the proposed role of asymmetric operator orientations in co-operative binding and transcription regulation can be explained by a proposing a two-stage oscillation model of CI dimer-operator complex formation: A CI dimer binds to an asymmetric operator in which each subunit bound to a DNA half-site alternatively switches between an extended- and a condensed- state with none state being favorable to interaction with a neighboring protein the other state being not. The oscillation frequency varies from operator to operator depending upon the binding strength of the corresponding NC half in each operator. (ii) Role of RNA in bacterial chromosome structure: From RNA deep sequencing data and of RIP-Chip assays of HU protein we found that out of a cluster of a typical DNA REP elements around the chromosome, one of them, REP325, encodes an RNA naR4 that participates in nucleoid architecture. The RNA is composed of a Y motif, an l motif and a Z2 motif. Furthermore, gel shift assays demonstrate that the transcript binds to HUaa and ab, but not to HUbb. Data from TEM analysis revealed that deletion of these repeats or of HU genes loosened nucleoid structure, which could be restored qualitatively by expression of naR4 RNA from a plasmid. In vitro data from AFM suggested that only HUaa and HUab could condense plasmid DNA in the presence of naR4 RNA. The presence of both motifs (Z2 and Y motif) in naR4 RNA is essential. In vivo data from 3C analysis confirmed that HU and/or naRNA participate in the intra-chromosomal interactions of REPs which are significantly affected by deletion of hupab and/or rep325. We propose a model for HU and naRNA mediated DNA condensation, in which HU molecules bind to different DNA sequences and DNA-bound HU proteins interact with each other through a na4RNA in a pair wise fashion. We believe the new family of non-coding RNA, nucleoid architectural naRNA plays roles in prokaryotic nucleoid architecture.
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