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

Murakami, Katsuhiko; Davydova, Elena K.; Rothman-Denes, Lucia B.

doi:10.1073/pnas.0712325105

Cited by 19 publications

(35 citation statements)

References 33 publications

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“…Both mutant enzymes show similar affinities (200 μM) for GTP and GDP as the initiating nucleotide. Notably, the affinity of the K437A and E557A enzymes for the initiating nucleotide is similar to that of T7 RNAP (Table 1), whose active site is superimposable with that of N4 vRNAP (27); however, T7 RNAP lacks residues equivalent to N4 vRNAP K437 and E557. The 15-fold decrease in k cat upon replacement of E557 by Ala highlights the relevance of E557 in catalysis of the N4 vRNAP.…”

Section: Resultsmentioning

confidence: 96%

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

Gleghorn

Davydova

Basu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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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 þ2 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. D NA-dependent RNA polymerases (RNAPs) transcribe DNA genetic information into RNA and play a central role in gene expression. RNAP catalyzes a nucleotidyl transfer reaction, which is initiated by the nucleophilic attack of an O3′ oxyanion at the RNA 3′ terminus to the α-phosphate (αP) of the incoming nucleotide, resulting in phosphodiester bond formation and release of pyrophosphate (PPi). Both single-subunit T7 phage-like RNAPs and the multisubunit cellular RNAPs possess two nucleotide-binding sites for loading the RNA 3′ end (P site) and the incoming NTP (N site) (1, 2). A two metal-ion catalytic mechanism has been proposed, as the enzyme possesses two divalent catalytic and nucleotide-binding metal cations chelated by two or three conserved Asp residues (3). The catalytic metal is a Lewis acid, coordinating the RNA 3′-OH lowering its pK a and facilitating the formation of the attacking oxyanion. The nucleotide-binding metal is coordinated by the triphosphate of the incoming nucleotide and stabilizes a pentacovalent phosphate intermediate during the reaction. Both metal ions are proposed to have octahedral coordination at physiological Mg 2þ concentrations (4). During transcript elongation, RNAP carries out the loading of a single nucleotide substrate at the N site followed by a nucleotidyl transfer reaction with the RNA 3′ end at the P site; this cycle is repeated as elongation proceeds. X-ray crystal structures of the single-subunit T7 phage RNAP (2, 5) have depicted the process of transcript elongation in detail and reveal a conformational change of the Fingers subdomain during substrate loading to the active site as also observed in the A family of DNA polymerases (DNAPs) (6, 7).Initiation is the only step in the entire transcription process where two nucleotide substrates are loaded at the active site followed by a nucleotidyl transfer reaction. Compared with elongation, the process of initiation has not been well characterized by X-ray crystallography. An X-ray crystal structure of T7 RNAP initiation complex...

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Section: Resultsmentioning

confidence: 96%

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

Gleghorn

Davydova

Basu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…It could be hypothesized that the unique features of N4-like phages, especially the presence of an encapsulated RNA polymerase (37), may allow them to tolerate and replicate at cold (or even freezing) temperatures and under saline (or hypersaline) conditions. Indeed, it has been previously reported that the RNA polymerase in E. coli phage N4 can tolerate high-salt conditions (38). Further studies on cultured N4-like phages are necessary to ascertain whether cold tolerance is a defining feature of this phage class.…”

Section: Resultsmentioning

confidence: 99%

Novel N4 Bacteriophages Prevail in the Cold Biosphere

Zhan

Buchan

Chen

2015

Appl Environ Microbiol

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bColiphage N4 is a lytic bacteriophage discovered nearly half a century ago, and it was considered to be a "genetic orphan" until very recently, when several additional N4-like phages were discovered to infect nonenteric bacterial hosts. Interest in this genus of phages is stimulated by their unique genetic features and propagation strategies. To better understand the ecology of N4-like phages, we investigated the diversity and geographic patterns of N4-like phages by examining 56 Chesapeake Bay viral communities, using a PCR-clone library approach targeting a diagnostic N4-like DNA polymerase gene. Many new lineages of N4-like phages were found in the bay, and their genotypes shift from the lower to the upper bay. Interestingly, signature sequences of N4-like phages were recovered only from winter month samples, when water temperatures were below 4°C. An analysis of existing metagenomic libraries from various aquatic environments supports the hypothesis that N4-like phages are most prolific in colder waters. In particular, a high number of N4-like phages were detected in Organic Lake, Antarctica, a cold and hypersaline system. The prevalence of N4-like phages in the cold biosphere suggests these viruses possess yet-to-be-determined mechanisms that facilitate lytic infections under cold conditions. A s the most abundant microbial form, viruses play important roles in shaping host population structures, mediating genetic exchange between hosts, and modulating trophic transfer in marine food webs (1-3). Marine viral metagenomic studies suggest that viruses encompass the largest genetic repertoire in the ocean (4, 5), yet the functions of ϳ70% of the putative genes identified in viral metagenomic studies remain unknown (6). Recently, several novel phages infecting dominant marine bacteria have been isolated from the ocean (7,8). Both cultivation techniques and molecular approaches suggest that a great many marine viruses await discovery.Bacteriophage N4 was first isolated from sewer waters using Escherichia coli as a host nearly half a century ago (9) and remained a "genetic orphan" for decades, as no other genetically similar phages were characterized. Phage N4 has several unique features with regard to its morphology, physiology, and genome that have made it a focus of study. It has a 70-nm isocahedral capsid, and its genome size is 70 kb. More remarkably, N4 is the only known phage that does not rely on host RNA polymerase for early transcription (10, 11). Instead, it contains a DNA-dependent virionencapsidated RNA polymerase (vRNAP), which is coinjected with viral DNA into its host and initiates transcription (12). The phage also exhibits a lysis-inhibited infection cycle and an extremely large burst size (ca. 3,000 phages per cell), suggestive of a novel mechanism of cell lysis regulation (13,14).The recent renewed interest in the ecology of N4 was prompted by the isolation from a coastal estuary of two new N4-like phages, which infect bacteria of the marine Roseobacter lineage (15). Roseobacters are a widely ...

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“…Mitochondrial and chloroplast RNAPs are unlikely to have a structural element that is homologous or functionally analogous to the T7 RNAP N-terminal domain, since the N-terminal domains of these enzymes are highly divergent (11), and the promoter sequences recognized directly by the mitochondrial and chloroplast RNAPs do not extend upstream of Ϫ9. However, it has been suggested that the YMt RNAP has a promoter recognition loop like that seen in T7 RNAP (12), and the recently described structure of N4 RNAP exhibits a promoter loop similar to that of T7 RNAP (13), although this RNAP uses a hairpin promoter (Fig. 1) that is very different in structure from the T7 promoter.…”

Section: S Cerevisiae Ymtmentioning

confidence: 99%

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Nayak

Guo

Sousa

2009

Journal of Biological Chemistry

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Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family. S. cerevisiae YMt2 and phage T7 RNAPs are both members of the single subunit RNAP family, but their promoter sequences are very different. The T7 promoter is a 21-nucleotide (nt) duplex that includes 4 nt downstream of the transcription start site and 17 nt upstream of ϩ1 (1). The promoters of the closely related T3 phage or the more distantly related Sp6 phage(2) are identical in size to the T7 promoter but differ in sequence. In contrast, the YMt promoter is only 8 nt in length and is therefore similar in size to chloroplast promoters or the mitochondrial promoters of plants and other fungi, which are typically 8 -10 nt in length although highly disparate in sequence (3, 4) (Fig. 1).The mechanisms of promoter recognition by the YMt and T7 RNAP also appear to be distinct. T7 RNAP, like many of the phage polymerases, binds, melts, and initiates transcription as single subunit enzyme without any accessory transcription factor (5). In contrast, the YMt RNAP requires a specific accessory factor (Mtf1 (yeast mitochondrial transcription factor)) to initiate transcription from its promoter (6). This seeming disparity may, however, obscure underlying mechanistic similarity. For example, it was originally suggested (6) that Mtf1 might be homologous and analogous to prokaryotic RNAP -factor, which would mean that Mtf1 contributes directly to promoter recognition, a mechanism distinct from that seen with T7 RNAP, where the polymerase contains all of the promoter recognition elements. However, subsequent work showed that YMt RNAP can initiate transcription without Mtf1 if the promoter is supercoiled or contains a heterduplex ("bubble") ...

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

Cited by 19 publications

References 33 publications

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

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

Novel N4 Bacteriophages Prevail in the Cold Biosphere

A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

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