T4 Transcription Research Articles

Self-Supplying Guide RNA-Mediated CRISPR/Cas12a Fluorescence System for Sensitive Detection of T4 PNKP.

Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better guidance in PNKP-related diagnostics and drug screening. Although the CRISPR/Cas12a system shows great promise in DNA-based signal amplification protocols, its guide RNAs with small molecular weight often suffer nuclease degradation during storage and utilization, resulting in reduced activation efficiency. Herein, we proposed a self-supplying guide RNA-mediated CRISPR/Cas12a system for the sensitive detection of T4 PNKP in cancer cells, in which multiple copies of guide RNA were generated by in situ transcription. In this assay, T4 PNKP was chosen as a model, and a dsDNA probe with T7 promoter region and the transcription region of guide RNA were involved. Under the action of T4 PNKP, the 5'-hydroxyl group of the dsDNA probe was converted to a phosphate group, which can be recognized and digested by Lambda Exo, resulting in dsDNA hydrolysis. The transcription template was destroyed, which resulted in the failure to generate guide RNA by the transcription pathway. Therefore, the CRISPR/Cas12a system could not be activated to effectively cleavage the F-Q-reporter, and the fluorescence signal was turned off. In the absence of T4 PNKP, the 5'-hydroxyl group of the substrate DNA cannot be digested by Lambda Exo. The intact dsDNA acts as the transcription template to generate a large amount of guide RNA. Finally, the formed Cas12a/gRNA complex triggered the reverse cleavage of Cas12a on the F-Q-reporter, resulting in a "turn-on" fluorescence signal. This strategy displayed sharp sensitivity of T4 PNKP with the limit of detection (LOD) down to 0.0017 mU/mL, which was mainly due to the multiple regulation effect of transcription amplification. In our system, the dsDNA simultaneously serves as the T4 PNKP substrate, transcription template, and Lambda Exo substrate, avoiding the need for multiple probe designs and saving costs. By integrating the target recognition, Lambda Exo activity, and trans-cleavage activity of Cas12a, CRISPR/Cas12a catalyzed the cleavage of fluorescent-labeled short-stranded DNA probes and enabled synergetic signal amplification for sensitive T4 PNKP detection. Furthermore, the T4 PNKP in cancer cells has been evaluated as a powerful tool for biomedical research and clinical diagnosis, proving a good practical application capacity.

Structural plasticity of T4 transcription co-activator gp33 revealed by a protease-resistant unfolded state

Gene 33 protein (gp33) is a transcriptional coactivator for late genes of the T4 bacteriophage. gp33 possesses a 5-helix bundle core, with unstructured N- and C-terminal regions that account for >50% of the protein sequence. It plays a unique role of interacting with host RNA polymerase, couples transcription with DNA replication, and plays the dual function as repressor and co-activator in phage transcription. Here, we identify protein structural plasticity as the molecular basis of the dual nature in gp33. We find that gp33 has the peculiar property of remaining protease insensitive in its urea-unfolded state. Using NMR studies with spectroscopic measurements, we propose that intra-protein interactions are replaced by protein-urea interactions in gp33. This process not only unfolds gp33 but also renders it protease-resistant. Our studies shed new light on the unique structural malleability of gp33 that might be important in its transition from a repressor to a late transcription co-activator.

Regulating phosphoenolpyruvate carboxylase activity by copper-induced expression method and exploring its role of carbon flux distribution in Synechocystis PCC 6803

The petE gene transcription can be regulated by copper titer in some cyanobacteria. In order to regulate the activity of phosphoenolpyruvate carboxylase (PEPC) in Synechocystis PCC 6803, “Ω-PpetE” fragment, containing T4 transcription terminating sequence, anti-spectinomycin gene and petE promoter sequence was inserted between the native promoter and the ORF of ppc (gene encoding PEPC). After several rounds of selection, a stable and completely segregated mutant was attained. Cultured in different copper titers, the mutant presented narrow enzyme activity variance in the late exponential phase. A basal enzyme activity was observed in the culture without copper. When copper titer was increased to more than 280 nM which was the normal level of BG11 broth, the activity was stable. When PEPC activity was downregulated, the content of intracellular total carbonhydrate increased significantly and the content of intracellular total protein decreased. In central metabolic network, regulation characteristics of PEPC in Synechocystis PCC 6803 showed considerable robustness. Within the studied range of regulation, it can be regarded a pivotal node in central metabolic network which controls carbon flux distribution and decides the ratio of major biomass constituents.

Crystallographic and NMR Analyses of UvsW and UvsW.1 from Bacteriophage T4

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3'-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7A and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases.

Mutational Analysis of σ 70 Region 4 Needed for Appropriation by the Bacteriophage T4 Transcription Factors AsiA and MotA

Transcriptional activation of bacteriophage T4 middle promoters requires σ 70-containing Escherichia coli RNA polymerase, the T4 activator MotA, and the T4 co-activator AsiA. T4 middle promoters contain the σ 70 –10 DNA element. However, these promoters lack the σ 70 –35 element, having instead a MotA box centered at –30, which is bound by MotA. Previous work has indicated that AsiA and MotA interact with region 4 of σ 70, the C-terminal portion that normally contacts –35 DNA and the β-flap structure in core. AsiA binding prevents the σ 70/β-flap and σ 70/–35 DNA interactions, inhibiting transcription from promoters that require a –35 element. To test the importance of residues within σ 70 region 4 for MotA and AsiA function, we investigated how σ 70 region 4 mutants interact with AsiA, MotA, and the β-flap and function in transcription assays in vitro. We find that alanine substitutions at residues 584–588 (region 4.2) do not impair the interaction of region 4 with the β-flap or MotA, but they eliminate the interaction with AsiA and prevent AsiA inhibition and MotA/AsiA activation. In contrast, alanine substitutions at 551-552, 554-555 (region 4.1) eliminate the region 4/β-flap interaction, significantly impair the AsiA/σ 70 interaction, and eliminate AsiA inhibition. However, the 4.1 mutant σ 70 is still fully competent for activation if both MotA and AsiA are present. A previous NMR structure shows AsiA binding to σ 70 region 4, dramatically distorting regions 4.1 and 4.2 and indirectly changing the conformation of the MotA interaction site at the σ 70 C terminus. Our analyses provide biochemical relevance for the σ 70 residues identified in the structure, indicate that the interaction of AsiA with σ 70 region 4.2 is crucial for activation, and support the idea that AsiA binding facilitates an interaction between MotA and the far C terminus of σ 70.

A Family of Anti-σ 70 Proteins in T4-type Phages and Bacteria that are Similar to AsiA, a Transcription Inhibitor and Co-activator of Bacteriophage T4

Anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic RNA polymerase. The bacteriophage T4 anti-sigma70 protein, AsiA, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of Escherichia coli RNA polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 DNA sequence. In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical sigma70 -10 consensus sequence but have a MotA box sequence centered at -30 rather than the sigma70 -35 sequence. The E.coli anti-sigma70 protein Rsd also interacts with region 4.2 of sigma70 and inhibits transcription from sigma70 promoters. Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to sigma70 region 4. We show that, like T4 AsiA, Rsd binds to sigma70 in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex. Previous work has assigned sigma70 amino acid F563, within region 4.1, as a critical determinant for AsiA binding. This residue is also involved in the binding of sigma70 to the beta-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between sigma70 region 4.1 and the beta-flap. We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with sigma70 region 4 is diminished by the substitution F563Y. We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from sigma70-dependent promoters. We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary sigma factors. In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA. We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA.

The bacteriophage T4 transcription activator MotA interacts with the far-C-terminal region of the sigma70 subunit of Escherichia coli RNA polymerase.

Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the sigma70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at -30, and also interacts with sigma70. We show here that the N-terminal half of MotA (MotA(NTD)), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of sigma70 with comparable sigma38 residues abolishes the interaction with MotA(NTD) in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a sigma70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA(CTD)) binds DNA with a K(D(app)) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of sigma70 helps to substitute functionally for an interaction between sigma70 and a promoter -35 element.

Solution structure of the constant region of nuclear envelope protein LAP2 reveals two LEM-domain structures: one binds BAF and the other binds DNA.

The nuclear envelope proteins LAP2, emerin and MAN1 share a conserved approximately 40-residue 'LEM' motif. Loss of emerin causes Emery-Dreifuss muscular dystrophy. We have solved the solution NMR structure of the constant region of human LAP2 (residues 1-168). Human LAP2(1-168) has two structurally independent, non-interacting domains located at residues 1-50 ('LAP2-N') and residues 111-152 (LEM-domain), connected by an approximately 60-residue flexible linker. The two domains are structurally homologous, comprising a helical turn followed by two helices connected by an 11-12-residue loop. This motif is shared by subdomains of T4 endonuclease VII and transcription factor rho, despite negligible (< or =15%) sequence identity. NMR chemical shift mapping demonstrated that the LEM-domain binds BAF (barrier-to-autointegration factor), whereas LAP2-N binds DNA. Both binding surfaces comprise helix 1, the N-terminus of helix 2 and the inter-helical loop. Binding selectivity is determined by the nature of the surface residues in these binding sites, which are predominantly positively charged for LAP2-N and hydrophobic for the LEM-domain. Thus, LEM and LEM-like motifs form a common structure that evolution has customized for binding to BAF or DNA.

ADP-ribosylation and early transcription regulation by bacteriophage T4.

Bacteriophage T4 codes at least for two ADP-ribosylating activities, the 76 kDa Alt and the 24 kDa Mod gene products. The main target for both enzymes is the host RNA polymerase. We cloned and sequenced the alt gene and overexpressed the corresponding enzyme. The recombinant protein shows ADP-ribosylating activities in vitro, as had been described earlier for the native enzyme isolated from phage heads. The native as well as the recombinant protein ADP-ribosylate the alpha-subunit of RNA polymerase, but also subunits beta, beta' and sigma 70 and perform an autoribosylation reaction. Taking advantage of the pKWIII test system, constructed to measure promoter strengths in vivo, it was found that ADP-ribosylation of RNA polymerase leads to an increase of transcription from T4 early promoters up to a factor of two. In an infected host cell this should cause an enhanced expression of T4 genes. Depending on whether RNA polymerase was ADP-ribosylated or not, it initiated transcription at T4 promoters with different sequence characteristics: unribosylated RNA polymerase recognizes the early T4 promoters by an extended -10 region, whereas the ribosylated enzyme selects for T4 early promoters with an extended T4-specific and highly conserved -35 region. These results may reflect how the virus, step by step imposes its genetic program on the host cell, and in part they give a rationale for the extension of the consensus sequence observed with these promoters. We also sequenced the genomic region of the T4 mod gene and found two open reading frames coding both for proteins of approximately 24 kDa. Up to now none of the reading frames could be cloned into E. coli in an active form, making it highly probable that the ADP-ribosylation pattern inflicted by gene product Mod on host RNA polymerase is deleterious to these bacteria. Comparisons of the amino acid sequences showed significant homologies among the two reading frames. Computer analysis reveals that both Mod sequences and also the sequence of the Alt protein exhibit a structural concordance with the catalytic domains of other prokaryotic ADP-mono-ribosyltransferases such as the Pseudomonas aeruginosa exotoxin A, the cholera labile enterotoxin, the diphteria toxin, the heat labile enterotoxin A of E. coli, and pertussis toxin. We present a detailed model for T4 transcription regulation.

A late exclusion of bacteriophage T4 can be suppressed by Escherichia coli GroEL or Rho.

A litCon mutation in Escherichia coli TU6 results in exclusion of bacteriophage T4 during the late, morphogenetic stage of its development at low temperatures. DNA was synthesized continuously in the infected cells, but less than 10% of the DNA made by 90 min after infection was packaged into DNAase-resistant particles, few viable phage were formed, and the cells lysed poorly. The exclusion could be relieved by conditions leading to elevated levels, determined immunologically, of the E. coli Rho protein (believed to be involved in regulation of T4 transcription), or chromosomally encoded E. coli GroEL (a chaperone known to be involved in phage assembly), or by supplying GroEL in trans from a plasmid. The two suppressing proteins appeared to act independently of each other. GroEL-suppression restored packaging to normal levels, perhaps by preventing GP23 from activating the host Lit protein; in addition DNA synthesis was delayed and reduced and cell lysis enhanced, demonstrating involvement of GroEL in both these processes. Rho suppression was less efficient. Since both transcription-termination-proficient and transcription-termination-deficient Rho suppressed, the results raise the possibility that Rho has a role during T4 development not directly involving transcription regulation.

Recombinant Escherichia coli RNA Polymerase: Purification of Individually Overexpressed Subunits and in Vitro Assembly

New improved methods were developed for the purification to apparent homogeneity of α,β,β′, and σ subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains. The purified subunits were assembled into enzymatically active RNAP holoenzyme (α2ββ′σ) using the optimal subunit molar ratio (α:β:β′:σ = 2:8:4:1) at a total protein concentration of 0.5 mg/ml. The presence of σ subunit and 10 μM ZnCl2 in the reconstitution mixture increased the yield of RNAP ∼ 4 times. The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56%. The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of [3H]UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h.

Bacteriophage T4 Alc protein: A transcription termination factor sensing local modification of DNA

Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E. coli. It is demonstrated that Alc acts as a site-specific termination factor. The Alc sites occur frequently in E. coli DNA, resulting in early cessation of elongation in several tested transcription units. Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site. Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination. Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA. In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc. Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term "memory" of recent pausing.

In vivo studies on the effects of immunity genes on early lytic transcription in the Halobacterium salinarium phage phi H.

We have studied in vivo the effects of putative immunity genes on the expression of an early lytic gene of the Halobacterium salinarium phage. phi H. We transformed an H. salinarium host with DNA coding for a putative repressor gene, the transcript of which has been designated T6. We show that, in vivo, this gene specifically shuts off production of the early lytic transcript T4. A construct carrying the DNA transcribed as T4, but without its putative repressor binding sequences, shows T4 transcription enhanced to a level comparable to that observed in lytic growth of mutant phages capable of growing on immune H. salinarium strains. This transcript is insensitive to the action of the T6 product. The product of this 'unrepressed' T4 transcript is able to complement in trans the repressed T4 on superinfecting phi H-sensitive phages, allowing these to grow on a strain containing the repressor gene. It has, however, no effect on the production of repressor. We also mapped the start and end points of two other transcripts, T9 and T10, which are expressed only in the lysogenic state by cells immune to superinfection by phage, cloned the coding DNA and used it to transform H. salinarium. This DNA, though transcribed by the transformants, has no detectable effect on the cells, which remain susceptible to phage infection.

Positive and Negative Transcriptional Elements of the Human Type IV Collagenase Gene

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

Positive and negative transcriptional elements of the human type IV collagenase gene.

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

The bacteriophage T4 gene mrh whose product inhibits late T4 gene expression in an Escherichia coli rpoH[formula omitted] mutant

In an Escherichia coli rpoH mutant (affecting σ 32 , heat-shock sigma factor) infected at high temperatures with wild-type T4 phage, late T4 transcription and consequently progeny production are dramatically impaired. This defect is due, in part, to insufficient activity of σ 70 [Frazier and Mosig, J. Bacteriol. 170 (1988) 1384–1388], which is necessary to indicate early T4 transcription. Unexpectedly, however, we found that, in this rpoH host, late T4 transcription is also impaired when the temperature is raised from 30 to 42°C late after infection, when T4 transcription is directed by the T4-encoded sigma factor, σ gp55 . Here, we show that a T4 gene that we call mrh (modulates rpoH), located at 14 kb on the T4 map, is responsible for the inhibition of late T4 transcription in the rpoH mutant host. T4 deletion mutants that lack the mrh gene can produce progeny in the rpoH host, but the Mrh protein, provided in trans from a plasmid-borne mrh gene, inhibits this growth. We have cloned and sequenced this T4 gene and synthesized the Mrh protein in a T7 RNA polymerase-dependent expression system. The M r of the Mrh protein deduced from the nucleotide sequence is 13419. Gene mrh is cotranscribed with several other, yet unidentified genes, both from an early promoter downstream from the late soc gene (encoding the small outer capsid protein) and from the late soc promoter further upstream. The mrh transcript initiated at the late promoter requires antitermination downstream from soc at a palindromic sequence that could fold into a stable hairpin containing a CUUCGG loop and that terminates most, if not all, transcripts initiated from early promoters further upstream [Macdonald and Mosig, EMBO J. 3(1984) 2863–2871].

Transcript Analyses of the uvsX-40-41 Region of Bacteriophage T4: Changes in the RNA as Infection Proceeds

The bacteriophage T4 genes uvsX (recombination protein), 40 (stimulates head formation), and 41 (DNA replication protein, part of the primase-helicase) are located together on the T4 genome (5'----3' uvsX-40-41). Previous analyses have indicated that all three proteins are expressed within 5 min after infection and that the level of 41 protein is less than that of uvsX. The mapping of transcripts from this region (reported here) shows that this expression arises from polycistronic messages detected between 2-4 min after infection, a time when phage-encoded factors are beginning to alter the host transcriptional apparatus. Major RNA 5' ends, 900 and 200 bases upstream of uvsX, show homology with previously deduced T4 transcription sites dependent on the T4 transcription factor motA (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258). Analysis of the 3' end of uvsX RNAs shows that initially most transcripts extend through gene 40 and 41, although approximately equal to one-fourth end just past uvsX (within gene 40). Later, more of the uvsX messages are monocistronic, having 5' ends close to the gene (200 and 55 bases upstream) and having the 3' end within gene 40. Thus, during infection the level of 41 RNA is lowered relative to uvsX message. Mapping of RNA expressed from an uvsX-40-41 plasmid in an uninfected cell gives 5' ends 700, 450, and 55 bases upstream of uvsX, i.e. positions different from those during T4 infection. This indicates that infection significantly changes the 5' ends for uvsX RNA, either by altering transcription initiation or RNA processing sites. In contrast, the majority of the uvsX RNAs expressed by plasmid in the uninfected cell do end at the stop mapped during infection. Thus, the host alone can produce this 3' end.

Roles of the Escherichia coli heat shock sigma factor 32 in early and late gene expression of bacteriophage T4.

We have analyzed early and late T4 gene expression at the levels of transcription and translation in rpoH+ (sigma 32+) and rpoH mutant cells infected under heat shock conditions. We found, as expected, that Escherichia coli cells must be adapted before infection to high temperature by the heat shock response to allow early T4 transcription, subsequent late gene expression, and progeny production at 42 degrees C. Unexpectedly, we found in addition that when rpoH mutant (sigma 32 mutant) cells were shifted from 30 to 42 degrees C 10 min after infection, late T4 genes were not expressed, even though DNA synthesis appeared to be normal.

Interactions of the bacteriophage T4 gene 55 product with Escherichia coli RNA polymerase. Competition with Escherichia coli sigma 70 and release from late T4 transcription complexes following initiation.

The bacteriophage T4 gene 55 protein endows Escherichia coli RNA polymerase core with the ability to recognize T4 late promoters. In these experiments, we show that T4 gene product (gp) 55, like the major E. coli RNA polymerase initiation subunit sigma 70, is released from elongating transcription complexes and is thus capable of cycling among molecules of RNA polymerase core. We also show that, paradoxically, sigma 70 is able to displace T4 gp55 from its site of binding to E. coli RNA polymerase core. After T4 infection, the core enzyme is ADP-ribosylated in each alpha subunit, and a T4-coded 11.4-kDa protein (rpbA) binds tightly to it; these modifications do not save gp55 from displacement by sigma 70. However, open T4 late promoter complexes, containing gp55, are resistant to attack by sigma 70.

Sequence organization and control of transcription in the bacteriophage T4 tRNA region

Bacteriophage T4 contains genes for eight transfer RNAs and two stable RNAs of unknown function. These are found in two clusters at 70 × 10 3 base-pairs on the T4 genetic map. To understand the control of transcription in this region we have completed the sequencing of 5000 base-pairs in this region. The sequence contains a part of gene 3, gene 1, gene 57, internal protein I, the tRNA genes and five open reading frames which most likely code for heretofore unidentified proteins. We have used subclones of the region to investigate the kinetics of transcription in vivo. The results show that transcription in this region consists of overlapping early, middle and late transcripts. Transcription is directed from two early promoters, one or two middle promoters and perhaps two late promoters. This region contains all of the features that are seen in T4 transcription and as such is a good place to study the phenomenon in more detail.