The bacteriophage N4 single-stranded DNA binding protein (N4SSB) is essential for phage replication, recombination and, unexpectedly, late transcription. We will use biochemical genetic and physical approaches to analyze the structure of N4SSB and its mechanism of activation at the bacteriophage N4 late promoters which are recognized by the E. coli sigma-70 RNA polymerase (RNAP). N4SSB is not a site- specific dsDNA binding protein. We have isolated N4SSB carboxy-terminal mutants, which are proficient for replication and recombination but deficient in supporting late transcription activation in vivo and in vitro. In contrast, mutants deficient in ssDNA binding do not support replication and recombination but activate transcription in vivo and invitro indicating that the ssDNA binding activity of transcription activations. Recent biochemical results indicate that N4SSB activates transcription by interacting directly with E. coli sigma-70 RNAP.

N4SSB activation-deficient mutants do not bind to RNAP. We propose to: 1) Identify the target of N4SSB interaction with E. coli RNAP by biochemical and genetic approaches. To that end we will determine the subunit which is the target of N4SSB in E. coli RNAP by affinity chromatography and crosslinking, and the binding constant between N4SSB and E. coli RNAP by fluorescence emmission anisotropy. We will isolate new RNAP mutants insensitive to N4SSB activation, and E. coli RNA polymerase suppressors of N4SSB activation- deficient mutants. We will characterize 397C RNAP and isolate new RNAP mutants in the C-terminus of the beta' subunit. 2) Characterize the activation domain of N4SSB. We will define biochemically the domain of N4SSB, clone and overproduced the domains, and determine the in vivo and in vitro effects of the N4SSB domains on transcription. 3) Determine promoter sequences required for N4SSB activation by studying the effect of N4SSB on the activity of E. coli RNAP at a set of well characterized promoters and identifying sequences at the N4 late promoters which are essential for N4SSB activation. 4) Determine the effect of N4SSB on promoter binding, open complex formation and promoter clearance to identify the steop in RNAP initiation which is the site of action of N4SSB. In addition, the contribution of supercoiling to these steps will be studied. 5) Characterize the role of E. coli RNAP associated factors in N4 late transcription. 6) Initiate structural studies of N4SSB. We expect that the proposed studies will provide new insights into the mechanism of transcription activation in prokaryotes as well as eukaryotes.

R01GM054431

1996-05-01

2001-04-30