T4 Late Gene Research Articles

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing.

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.

Transcription activation by a sliding clamp

Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.

Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the β-subunit flap domain of Escherichia coli RNA polymerase

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNA-mounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 Å-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the β-flap domain. Like domain 4 of the promoter specificity σ factor (σ(4)), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the β-flap domain. Nevertheless, gp33 and σ(4) are not structurally related. The gp33/β-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex.

Transcription of the T4 late genes

This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp of the T4 replisome. Gp45 becomes topologically linked to DNA through the action of its clamp-loader, but it is not site-specifically DNA-bound, as other transcriptional activators are. Gp45 facilitates RNA polymerase recruitment to late promoters by interacting with two phage-encoded polymerase subunits: gp33, the co-activator of T4 late transcription; and gp55, the T4 late promoter recognition protein. The emphasis of this account is on the sites and mechanisms of actions of these three proteins, and on their roles in the formation of transcription-ready open T4 late promoter complexes.

Dissection of the Bacteriophage T4 Late Promoter Complex

Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator is gp45, the viral replisome's sliding clamp; two sliding-clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) σ subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding-clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP–sliding clamp connections differ: only the gp33–gp45 linkage is essential for activation, while loss of the gp55–gp45 linkage impairs but does not abolish activation. Formation of transcription-ready promoter complexes by the sliding-clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55–gp45 but not the gp33–gp45 linkage. We interpret the relationship of these findings and our prior analysis to the common model of transcriptional initiation in bacteria in terms of two parallel pathways, with two RNAP holoenzymes and two DNA templates: (1) gp55–RNAP and the T4 late promoter execute basal transcription; (2) gp55–gp33–RNAP and the T4 late promoter with its mobile enhancer, the T4 sliding clamp, execute activated transcription. gp55 and gp33 perform σ-like functions, gp55 in promoter recognition and gp33 (as well as gp55) in enhancer recognition. gp33 operates the switch between these two pathways by repressing basal transcription.

Functional Role of the N-Terminal Domain of Bacteriophage T4-Gene Product 11

Bacteriophage T4 late gene product 11 (gp11), the three-dimensional structure of which has been solved by us to 2.0 A resolution, is a part of the virus' baseplate. The gp11 polypeptide chain consists of 219 amino acid residues and the functionally active protein is a three-domain homotrimer. In this work, we have studied the role of gp11 N-terminal domain in the formation of a functionally active trimer. Deletion variants of gp11 and monoclonal antibodies recognizing the native conformation of gp11 trimer have been selected. Long deletions up to a complete removal of the N-terminal domain, containing 64 residues, do not affect the gp11 trimerization, but considerably change the protein structure and lead to the loss of its ability to incorporate into the baseplate. However, the deletion of the first 17 N-terminal residues results in functionally active protein that can complete the 11(-)-defective phage particles in in vitro complementation assay. This region of the polypeptide chain is probably essential for gp11-gp10 stable complex formation at the early stages of phage baseplate assembly in vivo. A study of the gp10 deletion variants suggests that the central domain of gp10 trimer is responsible for the interaction with gp11.

The bacteriophage T4 late-transcription coactivator gp33 binds the flap domain of Escherichia coli RNA polymerase

Transcription of bacteriophage T4 late genes requires concomitant DNA replication. T4 late promoters, which consist of a single 8-bp -10 motif, are recognized by a holoenzyme containing Escherichia coli RNA polymerase core and the T4-encoded promoter specificity subunit, gp55. Initiation of transcription at these promoters by gp55-holoenzyme is inefficient, but is greatly activated by the DNA-loaded DNA polymerase sliding clamp, gp45, and the coactivator, gp33. We report that gp33 attaches to the flap domain of the Escherichia coli RNA polymerase beta-subunit and that this interaction is essential for activation. The beta-flap also mediates recognition of -35 promoter motifs by binding to sigma(70) domain 4. The results suggest that gp33 is an analogue of sigma(70) domain 4 and that gp55 and gp33 together constitute two parts of the T4 late sigma. We propose a model for the role of the gp45 sliding clamp in activation of T4 late-gene transcription.

The Mechanism of Transcriptional Activation by the Topologically DNA-linked Sliding Clamp of Bacteriophage T4

Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the σ-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer–template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation (∼320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 °C, but once opened at 30 °C remains open upon shift to the lower temperature. At a hybrid promoter for σ 70 and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33.

Role of Escherichia coli Hfq in late-gene silencing of bacteriophage T4 dmd mutant.

When the dmd gene of bacteriophage T4 is mutated, many T4 late genes are post-transcriptionally silenced because of rapid mRNA degradation. Here we show that the host hfq gene is involved in the rapid mRNA degradation in a dmd mutant. A disruption of the hfq gene caused weak but significant effects on the stability of late-gene mRNA, the late-gene expression and the growth of a dmd mutant. By probing with the soc gene, we found that disruption of the hfq gene impaired the translation-independent mRNA degradation, one of two mechanisms promoting rapid mRNA degradation. We also showed that purified Hfq protein bound stoichiometrically to soc RNA. These results strongly suggest that the hfq gene has a stimulatory role in dmd mutant-specific mRNA degradation.

Core-sigma interaction: probing the interaction of the bacteriophage T4 gene 55promoter recognition protein with E.coli RNA polymerase core

The bacterial RNA polymerase sigma subunits are key participants in the early steps of RNA synthesis, conferring specificity of promoter recognition, facilitating promoter opening and promoter clearance, and responding to diverse transcriptional regulators. The T4 gene 55 protein (gp55), the sigma protein of the bacteriophage T4 late genes, is one of the smallest and most divergent members of this family. Protein footprinting was used to identify segments of gp55 that become buried upon binding to RNA polymerase core, and are therefore likely to constitute its interface with the core enzyme. Site-directed mutagenesis in two parts of this contact surface generated gene 55 proteins that are defective in polymerase-binding to different degrees. Alignment with the sequences of the sigma proteins and with a recently determined structure of a large segment of sigma70 suggests that the gp55 counterpart of sigma70 regions 2.1 and 2.2 is involved in RNA polymerase core binding, and that sigma70 and gp55 may be structurally similar in this region. The diverse phenotypes of the mutants implicate this region of gp55 in multiple aspects of sigma function.

Dual targets of a transcriptional activator that tracks on DNA.

The sliding clamp of the bacteriophage T4 DNA polymerase, gp45, is also the proximal effector for activation of transcription of T4 late genes. We have identified the phage T4-encoded sigma factor gp55 and the co-activator gp33 as targets of gp45 in promoter complexes, and have shown that a conserved carboxy-terminal amino acid sequence of gp55 and gp33 is required for interaction with gp45. The respective contribution of each target-gp45 interaction to activation of transcription has been assessed by measuring promoter opening rates. The opening rate supported by interaction with both targets is far greater than the arithmetical sum of the separate contributions of each target, implying a synergistic activation of transcription through at least two separate interactions of the trimeric gp45.

A direct interaction between a DNA-tracking protein and a promoter recognition protein: implications for searching DNA sequence.

Bacteriophage T4 gene 45 protein, gp45, serves as the sliding clamp of viral DNA replication and as the activator of T4 late gene transcription. In the latter context, DNA tracking is an essential feature of the unique mechanism of action. T4 late promoters, which consist of a simple TATA box, TATAAATA, are recognized by the small sigma-family gene 55 protein, gp55, which binds to Escherichia coli RNA polymerase core. A direct and RNA polymerase-independent interaction of gp45 with gp55 has been demonstrated in two ways. (i) gp45 tracks along DNA; co-tracking of gp55 requires the previously documented DNA-loading process of gp45, and can be detected by photochemical crosslinking. (ii) The dynamics of DNA tracking by gp45 can be followed by footprinting; the catenated DNA-tracking state of gp45 is short-lived, but is stabilized by gp55. The ability of this topologically linked DNA-tracking transcriptional activator to interact directly with a promoter recognition protein suggests the existence of multiple pathways of promoter location, which are discussed.

Old phage, new insights: Two recently recognized mechanisms of transcriptional regulation in bacteriophage T4 development

The regulation of bacteriophage T4 middle and late gene expression involves previously unrecognized mechanisms. Middle transcription requires a DNA-binding transcriptional activator and a σ 70-binding co-activator. The coupling of late transcription to DNA replication is effected by a DNA-tracking protein that is loaded onto DNA by an assembly factor at enhancer-like entry sites. Late transcription also requires an RNA polymerase core-binding co-activator. The co-activators of T4 middle and late transcription share the property of depressing unactivated, basal transcription.

Old phage, new insights: two recently recognized mechanisms of transcriptional regulation in bacteriophage T4 development.

The regulation of bacteriophage T4 middle and late gene expression involves previously unrecognized mechanisms. Middle transcription requires a DNA-binding transcriptional activator and a sigma 70-binding co-activator. The coupling of late transcription to DNA replication is effected by a DNA-tracking protein that is loaded onto DNA by an assembly factor at enhancer-like entry sites. Late transcription also requires an RNA polymerase core-binding co-activator. The co-activators of T4 middle and late transcription share the property of depressing unactivated, basal transcription.

Fibritin Encoded by Bacteriophage T4 Gene wac has a Parallel Triple-stranded α-Helical Coiled-coil Structure

The bacteriophage T4 late gene wac (whiskers antigen c ontrol) encodes a fibrous protein which forms a collar/whiskers complex. Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin indicates a 90% α-helical content, and scanning calorimetry shows that the protein has several distinct domains. The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially occupied by apolar residue, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure. The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil. The α-helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues. Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the α-helices. Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature induced denaturation. Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard α-helical coiled-coil. Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles. The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo. We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of α-fibrous proteins.

Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gene 45 protein and late transcription.

We have used a molecular crowding reagent to define functions in the transcriptional activation of bacteriophage T4 late genes. This activation normally requires the three T4 DNA polymerase accessory proteins encoded by T4 genes 44, 62, and 45 (the gp44/62 complex and gp45), an enhancer-like cis-acting site, an RNA polymerase-bound coactivator, and an unobstructed path along the DNA joining the promoter to the enhancer. We show that molecular crowding eliminates the requirement for the gp44/62 complex and for the enhancer, retains the requirement for gp45 and its coactivator, and generates activated promoter complexes with nearly unchanged DNase I footprints. These experiments identify gp45 as the direct activator of transcription, and the gp44/62 complex as the assembly factor for gp45. They suggest that the enhancer serves as the normal, but not invariably essential, entry site for the gp45 DNA-tracking protein.

Transactivation of a plasmid-borne bacteriophage T4 late gene.

We examined how a plasmid-borne T4 late gene is activated by infecting T4 phage (transactivation). A gene fusion system was developed where expression of a late gene promoter fused to the lacZ gene may easily be followed by measuring beta-galactosidase activity. Considerable transactivation can occur, provided that the infecting phage contains a mutation which abolishes the denB-encoded endonuclease, and that the gene 46-encoded exonuclease is functional. The level of transactivation was correlated with the formation of high molecular weight DNA composed of tandem repeats of plasmid DNA. The formation of these molecules and subsequent transactivation depended on DNA replication and homology between phage and plasmid DNAs. Also the capacity of bacteriophage T4, grown on cells containing a plasmid-borne T4 gene, to transduce the plasmid provided indirect evidence of the formation of these tandem-repeat molecules. A good correlation was established between the ability to transduce and the presence of sequence homology between the phage and the plasmid. However, the requirement for phage/plasmid homology is no longer prerequisite if transcription from the plasmid is permitted by introducing an alc mutation into the infecting phage, presumably because this allows DNA replication to start through RNA priming.

A Transcriptional Enhancer Whose Function Imposes a Requirement That Proteins Track Along DNA

Transcriptional regulation of the bacteriophage T4 late genes requires the participation of three DNA polymerase accessory proteins that are encoded by T4 genes 44, 62, and 45, and that act at an enhancer-like site. Transcriptional activation by these DNA replication proteins also requires the function of an RNA polymerase-bound coactivator protein that is encoded by T4 gene 33 and a promoter recognition protein that is encoded by T4 gene 55. Transcriptional activation in DNA constructs, in which the enhancer and a T4 late promoter can be segregated on two rings of a DNA catenane, has now been analyzed. The ability of an interposed DNA-binding protein to affect communication between the enhancer and the promoter was also examined. Together, these experiments demonstrate that this transcription-activating signal is conveyed between its enhancer and a T4 late promoter by a DNA-tracking mechanism. Alternative activation mechanisms relying entirely on through-space interactions of enhancer-bound and promoter-bound proteins are excluded.

Activation and quantitative estimation of bacteriophage T4 late regulatory signal in cis- and transconditions

Activation of the bacteriophage T4 late gene 22 carried on the phage genome (ciscondition) or borne on a plasmid (transcondition) was quantitatively analyzed after phage infection using a lacZ reporter gene. Transcription from the late promoter was activated postreplicatively by concomitantly infecting T4 phage. A truncated T4-lacZ gene including only 15 bp of sequence upstream of the late promoter consensus of gene 22 TATAAATA, was fully activated in the ciscondition but induced to a reduced extent in the transcondition. A possible mechanism for trans-activation is discussed.

TheWacGene Product of Bacteriophage T4 Contains Coiled-Coil Structural Patterns

The bacteriophage T4 late gene wac (whisker antigen control) encodes the protein which forms the fibrous structure on the neck of the virion called whiskers. Amino acid sequence analysis of wac gene product, as deduced from the nucleotide sequence, indicate ten alpha-helical domains (19-40 residues long) with coiled-coil structural patterns. These regions comprise about 70% of the entire 486 amino acid sequence. The alpha-helices are separated by short stretches of polypeptide chain which are similar to the loop regions of the globular protein sequences. We propose a structural model for the dimer of wac gene product molecule, that we call fibritin in which two polypeptide chains associate in a parallel fashion and form a segmented alpha-helical coiled-coil rod similar to epidermal keratins.