Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis.

Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8 Šresolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P212121, with unit-cell parameters a = 53.7, b = 93.6, c = 124.9 Å, α = ß = γ = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.

Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases.

Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

Watching the bacteriophage N4 RNA polymerase transcription by time-dependent soak-trigger-freeze X-ray crystallography.

The challenge for structural biology is to understand atomic-level macromolecular motions during enzymatic reaction. X-ray crystallography can reveal high resolution structures; however, one perceived limitation is that it reveals only static views. Here we use time-dependent soak-trigger-freeze X-ray crystallography, namely, soaking nucleotide and divalent metal into the bacteriophage RNA polymerase (RNAP)-promoter DNA complex crystals to trigger the nucleotidyl transfer reaction and freezing crystals at different time points, to capture real-time intermediates in the pathway of transcription. In each crystal structure, different intensities and shapes of electron density maps corresponding to the nucleotide and metal were revealed at the RNAP active site which allow watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. Our study provides the temporal order of substrate assembly and metal co-factor binding at the active site of enzyme which completes our understanding of the two-metal-ion mechanism and fidelity mechanism in single-subunit RNAPs. The nucleotide-binding metal (Me(B)) is coordinated at the active site prior to the catalytic metal (Me(A)). Me(A) coordination is only temporal, established just before and dissociated immediately after phosphodiester bond formation. We captured these elusive intermediates exploiting the slow enzymatic reaction in crystallo. These results demonstrate that the simple time-dependent soak-trigger-freeze X-ray crystallography offers a direct means for monitoring enzymatic reactions.

Time-resolved events on the reaction pathway of transcript initiation by a single-subunit RNA polymerase: Raman crystallographic evidence.

The nucleotidyl transfer reaction leading to formation of the first phosphodiester bond has been followed in real time by Raman microscopy, as it proceeds in single crystals of the N4 phage virion RNA polymerase (RNAP). The reaction is initiated by soaking nucleoside triphosphate (NTP) substrates and divalent cations into the RNAP and promoter DNA complex crystal, where the phosphodiester bond formation is completed in about 40 min. This slow reaction allowed us to monitor the changes of the RNAP and DNA conformations as well as bindings of substrate and metal through Raman spectra taken every 5 min. Recently published snapshot X-ray crystal structures along the same reaction pathway assisted the spectroscopic assignments of changes in the enzyme and DNA, while isotopically labeled NTP substrates allowed differentiation of the Raman spectra of bases in substrates and DNA. We observed that substrates are bound at 2-7 min after soaking is commenced, the O-helix completes its conformational change, and binding of both divalent metals required for catalysis in the active site changes the conformation of the ribose triphosphate at position +1. These are followed by a slower decrease of NTP triphosphate groups due to phosphodiester bond formation that reaches completion at about 15 min and even slower complete release of the divalent metals at about 40 min. We have also shown that the O-helix movement can be driven by substrate binding only. The kinetics of the in crystallo nucleotidyl transfer reaction revealed in this study suggest that soaking the substrate and metal into the RNAP-DNA complex crystal for a few minutes generates novel and uncharacterized intermediates for future X-ray and spectroscopic analysis.

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.

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 approximately 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.

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.

Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4.

Bacteriophage N4 encapsidates a 3500-aa-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryoelectron microscopy. The virion has an icosahedral capsid with T=9 quasi-symmetry that encapsidates well-organized double-stranded DNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, 12 appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insight into its assembly and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system.

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 (K(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.

Coliphage N4 N-acetylmuramidase defines a new family of murein hydrolases.

Escherichia coli phage N4 infection leads to delayed host cell lysis, 3000 particles per infected bacterium and a small plaque phenotype. We show that bacteriophage N4 encodes a murein hydrolase (gp61) that is essential for N4 plaque-forming ability. gp61 has a high level of sequence similarity to hypothetical proteobacterial proteins, and Vibrio harveyi phage VHML ORF 19. Nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS) analysis of muropeptides from purified gp61 digestion of E. coli peptidoglycan indicates that gp61 is an N-acetylmuramidase. The EGGY motif present near the N terminus of gp61 and its homologs contains the glutamic acid residue essential for enzymatic activity. These results provide evidence that N4 gp61 and its homologs define a new family of N-acetylmuramidases (pfam05838.4, DUF847, COG3926). In contrast to its homologs, gp61 contains an N-terminal signal sequence. When expressed at levels present during phage infection, gp61 localizes primarily to the cell inner membrane; in contrast, over-expression of recombinant N4 gp61 is sufficient for rapid cell lysis. Overproduction of the recombinant Salmonella typhimurium (STM0016) homolog is sufficient for cell lysis only when fused to the gp61 N-terminal signal sequence. The results of subcellular localization and of mutagenesis of the gp61 N-terminal signal sequence indicate that gp61 must be released from the inner membrane to be catalytically active.

Phage N4 RNA polymerase II recruitment to DNA by a single-stranded DNA-binding protein.

Transcription of bacteriophage N4 middle genes is carried out by a phage-coded, heterodimeric RNA polymerase (N4 RNAPII), which belongs to the family of T7-like RNA polymerases. In contrast to phage T7-RNAP, N4 RNAPII displays no activity on double-stranded templates and low activity on single-stranded templates. In vivo, at least one additional N4-coded protein (p17) is required for N4 middle transcription. We show that N4 ORF2 encodes p17 (gp2). Characterization of purified gp2revealed that it is a single-stranded DNA-binding protein that activates N4 RNAPII transcription on single-stranded DNA templates through specific interaction with N4 RNAPII. On the basis of the properties of the proteins involved in N4 RNAPII transcription and of middle promoters, we propose a model for N4 RNAPII promoter recognition, in which gp2plays two roles, stabilization of a single-stranded region at the promoter and recruitment of N4 RNAPII through gp2-N4 RNAPII interactions. Furthermore, we discuss our results in the context of transcription initiation by mitochondrial RNA polymerases.

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.

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.

N4 RNA polymerase II, a heterodimeric RNA polymerase with homology to the single-subunit family of RNA polymerases.

Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.