Coliphage N4 transcription is carried out by three different RNA polymerases (RNAP) providing unique opportunities to study structure-function relationships in RNAPases, the role of DNA structure, supercoiling and single-stranded DNA binding (SSB) proteins in transcriptional activation. We propose to continue studies on the structure, mechanism of promoter recognition and activation of the virion-encapsidated, 320-kDa single-polypeptide RNAP (vRNAP) which is responsible for the synthesis of phage early RNAs. vRNAP recognizes a 5-7 bp stem, 3b loop hairpin and specific sequences at its promoters. N4 vRNAP promoters are activated by supercoiling and Eco SSB. Supercoiling leads to a Mg(II)- and sequence-dependent extrusion of the promoter hairpins. We have proposed that Eco SSB invades this structure with melting of the complementary strand hairpin and stabilization of the template strand hairpin which is recognized by vRNAP. We propose to characterize the DNA structural transition occurring at vRNAP promoters using fluorescence resonance transfer (FRET). We will further characterize the interaction vRNAP with its promoters by elucidating the sequence determinants of promoter binding through a binding site selection protocol and the use of a quantitative binding assay. The role of Eco SSB in providing the structure of the """"""""activated promoter"""""""" will be defined by footprinting mapping of the topology of the """"""""activated"""""""" promoter, determining the role of promoter downstream sequences and of single-strandedness in EcoSSB invasion and footprinting of vRNAP at the """"""""activated promoter"""""""". The stoichiometry of Eco SSB binding at the activated promoter will be determined by scanning force microscopy. We will characterize the role of Eco SSB and promoter sequences in promoter clearance. Finally, we will define the vRNAP domain involved in promoter recognition through proteolysis and crosslinking studies. Results of these experiments will provide insights into a new strategy of promoter-RNAP interaction, novel determinants of protein-DNA interaction, as well as the role of single-stranded DNA binding proteins in regulation of gene expression.
Showing the most recent 10 out of 41 publications
