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Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection. The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs. In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured. We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription. We report here that Escherichia coil single-stranded DNA-binding protein (SSB) is also required for N4 early transcription. In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription. The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation. The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute. Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription. The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.[Key Words: Coliphage N4; RNA polymerase; single-stranded DNA-binding protein; transcriptional activation; template supercoiling] Bacteriophage N4 is a double-stranded DNA virus specific for Escherichia coli K 12 strains. Transcription of its genome is regulated through the sequential activity of three distinct RNA polymerases (for review, see Kiino and Rothman-Denes 1988). Early N4 transcripts are synthesized by a rifampicin-resistant, N4-coded RNA polymerase, which is carried within the virion and injected into the cell at the start of infection (Falco et al. 1977). N4 middle transcripts are synthesized by a second N4-coded RNA polymerase (N4 RNA polymerase II) (Zehring and Rothman-Denes 1983; . Late in infection, the N4-coded, single-stranded DNA-binding protein directs the host RNA polymerase to transcribe the N4 structural genes (Zivin et al. 1981; N. Baek, M. Choi and L. Rothman-Denes, in prep.) Purified virion RNA polymerase shows peculiar template specificity. It is unable to use native duplex genomic N4 DNA as a template (Falco et al. 1978(Falco et al. , 1980 polymerases, it transcribes denatured or single-stranded, promoter-containing templates accurately and efficiently (Haynes and Rothman-Denes 1985; Glucksmann et al. 1992). This result suggests that the structure of N4 DNA must be altered upon entry into the cell to allow transcription by the virion RNA polymerase. It is likely that the N4 virion RNA polymerase utilizes a supercoiled template because active host DNA gyrase is required for N4 early RNA synthesis (Falco et al. 1978). However, in vitro, supercoiled, promoter-containing templates do not support virion RNA polymeras...

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