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Coliphage N4 virion RNA polymerase (vRNAP), which is injected into the host upon infection, transcribes the phage early genes from promoters that have a 5-bp stem-3 nt loop hairpin structure. Here, we describe the 2.0-[Angstrom] resolution x-ray crystal structure of N4 mini-vRNAP, a member of the T7-like, single-unit RNAP family and the minimal component having all RNAP functions of the full-length vRNAP. The structure resembles a 'fisted right hand' with Fingers, Palm and Thumb subdomains connected to an N-terminal domain. We established that the specificity loop extending from the Fingers along with W129 of the N-terminal domain play critical roles in hairpin-promoter recognition. A comparison with the structure of the T7 RNAP initiation complex reveals that the pathway of the DNA to the active site is blocked in the apo-form vRNAP, indicating that vRNAP must undergo a large-scale conformational change upon promoter DNA binding and explaining the highly restricted promoter specificity of vRNAP that is essential for phage early transcription.

Bacteriophage N4 minivirion RNA polymerase (mini-vRNAP), the RNA polymerase (RNAP) domain of vRNAP, is a member of the T7-1ike RNAP family. Mini-vRNAP recognizes promoters that comprise conserved sequences and a 3-base loop-5-base pair (bp) stem DNA hairpin structure on single-stranded templates. Here, we defined the DNA structural and sequence requirements for mini-vRNAP promoter recognition. Mini-vBNAP binds a 20-nucleotide (nt) N4 P2 promoter deoxyoligonucleotide with high affinity ([K.sub.d] = 2 nM) to form a salt-resistant complex. We show that mini-vRNAP interacts specifically with the central base of the hairpin loop (-11G) and a base at the stem (-8G) and that the guanine 6-keto and 7-imino groups at both positions are essential for binding and complex salt resistance. The major determinant (-11G), which must be presented to mini-vRNAP in the context of a hairpin loop, appears to interact with mini-vRNAP Trp-129. This interaction requires template single-strandedness at positions -2 and -1. Contacts with the promoter are disrupted when the RNA product becomes 11-12 nt long. This detailed description of vRNAP interaction with its promoter hairpin provides insights into RNAP-promoter interactions and explains how the injected vRNAP, which is present in one or two copies, recognizes its promoters on a single copy of the injected genome. T7-like RNA polymerase

Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage genome. Escherichia coli single-stranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on supercoiled DNA templates containing vRNAP promoters. In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded, promoter-containing templates with in vivo specificity; however, the RNA product is not displaced, thus limiting template usage to one round. We show that EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling. EcoSSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated EcoSSB mutant proteins, human mitochondrial SSB (Hsmt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hsmt-EcoSSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of EcoSSB. T7 RNAP contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme. No sequence similarity to this domain exists in vRNAP. Hereby, we propose a unique role for EcoSSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding.

We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction. [ABSTRACT FROM AUTHOR]