Elena K. Davydova

The phage N4 virion RNA polymerase catalytic domain is related to single‐subunit RNA polymerases

In vitro, bacteriophage N4 virion RNA polymerase (vRNAP) recognizes in vivo sites of transcription initiation on single‐stranded templates. N4 vRNAP promoters are comprised of a hairpin structure and conserved sequences. Here, we show that vRNAP consists of a single 3500 amino acid polypeptide, and we define and characterize a transcriptionally active 1106 amino acid domain (mini‐vRNAP). Biochemical and genetic characterization of this domain indicates that, despite its peculiar promoter specificity and lack of extensive sequence similarity to other DNA‐dependent RNA polymerases, mini‐vRNAP is related to the family of T7‐like RNA polymerases.

Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase

Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion 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 super-coiled 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.

Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

Bacteriophage N4 minivirion RNA polymerase (mini-vRNAP), the RNA polymerase (RNAP) domain of vRNAP, is a member of the T7-like 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-vRNAP binds a 20-nucleotide (nt) N4 P2 promoter deoxyoligonucleotide with high affinity (Kd = 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.

X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase

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-Å 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.

X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides

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.

Bacteriophage N4-coded, virion-encapsulated DNA-dependent RNA polymerase.

Publisher Summary This chapter reviews the bacteriophage N4 vRNAP and its unique ability to transcribe promoter-containing single-stranded DNAs with specificity, providing a tool for the synthesis of RNAs from synthetic oligonucleotide templates or from templates cloned in M13 DNA. It is an ideal enzyme for the synthesis of short RNAs in large amounts at limiting template concentrations in the presence of Eco SSB, for several reasons. N4 vRNAP displays high fidelity and, in contrast to T7 RNAP, does not appear to produce abortive products. It discusses the length of the transcripts that can be synthesized by mini-vRNAP is limited by the length of the available single-stranded DNA template. vRNAP is useful for the synthesis of specific RNA–DNA hybrids when transcription is carried out in the absence of Eco SSB. The chapter explores the discovery of mini-vRNAP that has provided a useful tool to study polymerase–promoter, polymerase–substrate, and polymerase–product interactions. Finally, identification of the vRNAP transcriptionally active domain raises questions on the possible roles of the amino and carboxy-terminal domains of the polypeptide. Experiments in progress have indicated roles for these domains in virion morphogenesis, and the initial steps of phage infection.

Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes

Bacteriophage N4 mini-virion RNA polymerase (mini-vRNAP), the 1106-amino acid transcriptionally active domain of vRNAP, recognizes single-stranded DNA template-containing promoters composed of conserved sequences and a 3-base loop–5-base pair stem hairpin structure. The major promoter recognition determinants are a purine located at the center of the hairpin loop (–11G) and a base at the hairpin stem (–8G). Mini-vRNAP is an evolutionarily highly diverged member of the T7 family of RNAPs. A two-plasmid system was developed to measure the in vivo activity of mutant mini-vRNAP enzymes. Five mini-vRNAP derivatives, each containing a pair of cysteine residues separated by ∼100 amino acids and single cysteine-containing enzymes, were generated. These reagents were used to determine the smallest catalytically active polypeptide and to map promoter, substrate, and RNA-DNA hybrid contact sites to single amino acid residues in the enzyme by using end-labeled 5-iododeoxyuridine- and azidophenacyl-substituted oligonucleotides, cross-linkable derivatives of the initiating nucleotide, and RNA products with 5-iodouridine incorporated at specific positions. Localization of functionally important amino acid residues in the recently determined crystal structures of apomini-vRNAP and the mini-vRNAP-promoter complex and comparison with the crystal structures of the T7 RNAP initiation and elongation complexes allowed us to predict major rearrangements in mini-vRNAP in the transition from transcription initiation to elongation similar to those observed in T7 RNAP, a task otherwise precluded by the lack of sequence homology between N4 mini-vRNAP and T7 RNAP.