Prereplicative Genes Research Articles

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity.

Identification of two middle promoters upstream DNA ligase gene 30 of bacteriophage T4

Bacteriophage T4 DNA ligase gene 30 lies in the cluster of prereplicative genes located counterclockwise from map units 149 to 121. Based on the early transcription studies this gene has been considered as a typical early gene of bacteriophage T4. In agreement with this assignment, two strong T4 early promoters, P E 30.8 (128.6) and P E 30.7 (128.2), located about 3.1 and 2.7 kb upstream from gene 30 have been revealed by promoter mapping and sequence analysis. In addition, the existence of a putative early promoter just upstream of gene 30 was proposed from the sequence data. However, here we show that the putative early promoter just upstream of gene 30 is, in fact, a T4 middle promoter. Furthermore, we detected one more middle promoter located in the genomic region between early promoter P E 30.7 (128.2) and DNA ligase gene 30 in the coding region of gene 30.3. Both new middle promoters have differences from the consensus MotA box, while their −10 regions match the σ 70 consensus sequence very well. The 5′ ends of MotA-dependent transcripts directed from these promoters, as well as the kinetics of 5′ end accumulation in the cells, have been determined by primer extension analysis. The results of these analyses indicate that both MotA-dependent and MotA-independent promoters control the transcription of T4 DNA ligase gene 30 in vivo. Moreover, we show that the first transcripts for gene 30 are directed from its own middle promoter, P M 30.

Endoribonuclease RegB from bacteriophage T4 is necessary for the degradation of early but not middle or late mRNAs

The RegB endoribonuclease from bacteriophage T4 cleaves early mRNAs specifically in the middle of the sequence GGAG. We show here that RegB is required for the degradation of bulk T4 early mRNA. In the absence of RegB, the chemical half-life of early transcripts is increased nearly fourfold, whereas their functional half-life is increased twofold. RegB also regulates the translation of several prereplicative genes. The synthesis of several early proteins is down-regulated, probably as a consequence of RegB cleavages in the Shine-Dalgarno sequence of these genes. The synthesis of several other proteins is up-regulated, suggesting that processing by RegB might improve translation by changing the conformation of a transcript. In contrast, RegB does not affect the average half-life of middle and late mRNA. An analysis of the susceptibility to RegB of many GGAG motifs carried by these mRNA species showed that most middle and all late GGAG-carrying mRNAs escape RegB processing in spite of the fact that the enzyme is acting at least until ten minutes post-infection. The sensitivity or resistance to RegB observed during phage infection could be reproduced in uninfected Escherichia coli cells and in vitro. This shows that the GGAG-carrying RNAs that are uncut during T4 infection are not substrates, whatever the period of the T4 cycle when the transcripts are made.

Characterisation of the bacteriophage T4 comCα55.6 and comCJ mutants. A possible role in an antitermination process

We have performed a new screen for T4 mutants ( comC) that overcome the phage growth restriction caused by the Escherichia coli rholtabC mutants. We show that one such mutant ( comCJ) identifies a different gene from that identified by canonical comC mutants. We compare the regulation of T4 prereplicative transcription in a rholtabC mutant infected by T4 wild-type, by a canonical comC mutant ( comCα55.6) and by comCJ. The transcription rates of the two prereplicative genes 39 and 43 is depressed in a T4 wild-type infected tabC host mutant. When comCα55.6 and/or comCJ single and double mutants are the infecting phages, transcription of genes 39 and 43 is resumed to different extents; in particular, in the double mutant infections there appears to be a synergistic effect on transcription. Furthermore, we find that the comCα55.6 phage mutant affects the transcription rate of the gene rIIA in a wild-type host.

Characterisation of the bacteriophage T4 comC alpha 55.6 and comCJ mutants. A possible role in an antitermination process.

We have performed a new screen for T4 mutants (comC) that overcome the phage growth restriction caused by the Escherichia coli rho/tabC mutants. We show that one such mutant (comCJ) identifies a different gene from that identified by canonical comC mutants. We compare the regulation of T4 prereplicative transcription in a rho/tabC mutant infected by T4 wild-type, by a canonical comC mutant (comC alpha 55.6) and by comCJ. The transcription rates of the two prereplicative genes 39 and 43 is depressed in a T4 wild-type infected tabC host mutant. When comC alpha 55.6 and/or comCJ single and double mutants are the infecting phages, transcription of genes 39 and 43 is resumed to different extents; in particular, in the double mutant infections there appears to be a synergistic effect on transcription. Furthermore, we find that the comC alpha 55.6 phage mutant affects the transcription rate of the gene rIIA in a wild-type host.

New control elements of bacteriophage T4 pre-replicative transcription

Bacteriophage T4 pre-replicative genes are transcribed, by Escherichia coli RNA polymerase, in two alternative modes: an early mode and a middle mode. Middle mode transcription is under the control of at least one viral protein, p motA. We have identified two additional viral genes, motB and motC, that map in the dispensable region of the T4 genome, between genes 39 and 56. p motB and p motC are diffusible factors which provide an alternative to the motA dependent mode of middle transcription of many T4 genes. Deletions of motB and motC are in fact lethal only in combination with a motA mutant. motB controls one of the alternative modes of transcription of the rIIA gene. When motA or motB are missing, transcription of rIIA is quantitatively unaffected; when both are missing the transcription rate drops by about 75%. Control of transcription of the tRNA gene cluster is more complex. Transcription of subcluster 2 is maximally reduced (70%) only by deletions that, besides motB, cut out an adjacent region. We guess that this adjacent region codes for an additional control element, which we call motC. The motB gene is situated in a 750-base region between the left end-points of del(39–56)-1 and -4.

Partial characterization of the mitogenic action of pp60v-src, the oncogenic protein product of the src gene of avian sarcoma virus.

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, are transformed at 36 degrees C, but at 40 degrees C they behave as nontransformed cells because of the inactivation of the abnormally thermolabile pp60v-src product of the virus' transforming src gene. At 40 degrees C, these tsLA23-NRK cells were arrested in G1/G0 by severe serum deprivation. They were induced to enter G1, initiate DNA synthesis 7 or 10 hours later, and then divide as (1) nontransformed cells by adding serum or platelet-derived growth factor (PDGF) at 40 degrees C, or (2) transformed cells by lowering the temperature to a pp60v-src-activating 36 degrees C without adding exogenous growth factor(s). The level of pp60v-src kinase activity rose dramatically in these serum-deprived cells within 30 minutes of lowering the temperature to the permissive 36 degrees C, and it fell just as rapidly when the cells were returned to the restrictive 40 degrees C. As little as a 2-hour exposure to 36 degrees C, with an attendant 2-hour burst of pp60v-src kinase activity, was enough to stimulate serum-deprived tsLA23-NRK cells to transit G1 and initiate DNA replication, but not to divide. Much more prolonged pp60v-src activity was needed for these serum-deprived cells to complete their cycle and divide. The prereplicative development of quiescent tsLA23-NRK cells stimulated by serum or PDGF was accompanied by greatly increased protein synthesis and slightly decreased protein degradation, but the pp60v-src-stimulated cells progressed through G1 and initiated DNA replication without appreciably affecting the protein synthetic machinery of the cell. The cells stimulated by the mitogenic action of pp60v-src, like the cells stimulated by serum, needed to activate early prereplicative genes in order to initiate DNA replication. The needed RNA transcripts induced by serum and pp60v-src were produced with comparable efficiency, although it took longer for pp60v-src-stimulated cells to translate these transcripts and to initiate DNA replication, probably because of their unstimulated protein-synthetic machinery.

Host-virus interactions in the control of T4 prereplicative transcription: II. Interaction between tabC ( rho) mutants and T4 mot mutants

In this paper we describe bacterial mutants ( tabG) that reinforce the effect of T4 mot mutants (Mattson et al., 1978) tsG1 and amG1. tabG mutants map close to or in the Escherichia coli RNA polymerase rpoB gene. We explore the effect of mot-deficient T4 infections in tabC mutants (Caruso et al., 1979). We find that: (1) in such infections the synthesis of most T4 proteins of molecular weight greater than 24,000 is inhibited; (2) T4-specific transcription is largely restricted to those species that have been defined as immediate early. We conclude that most tabC bacterial mutants affect transcription of T4 prereplicative genes and confirm that T4 mot mutants also affect transcription of T4 prereplicative genes. We discuss a model where many (most) T4 prereplicative genes are transcribed in two alternative modes: from a rho ( tabC)-sensitive, mot-independent early promoter and from a tabC-insensitive, mot-dependent, middle promoter.

Level of specific prereplicative mRNA's during bacteriophage T4 regA-, 43- and T4 43- infection of Escherichia coli B

The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation.

Synthesis of functional bacteriophage T4-delayed early mRNA in the absence of protein synthesis

When Escherichia coli B207 is grown either aerobically or under limited aerobic conditions, pretreated with chloramphenicol to block protein synthesis, and then infected with bacteriophage T4, the phage RNA which accumulates, termed "immediately early" (IE), contains the transcripts of a limited number of prereplicative genes. Among the transcripts which accumulate is the mRNA which serves as a template for deoxycytidylate hydroxymethylase (HMase) synthesis. Among the prereplicative gene transcripts which do not accumulate under these conditions are deoxycytidine triphosphatase (dCTPase), alpha-glucosyl transferase (alphg-gt), and deoxynucleotide kinase (kinase); these genes have been termed "delayed early" (DE). In contrast, when protein synthesis is inhibited by depleting aerobically grown E. coli B207 of K+, both IE and DE T4 RNA accumulate, but these transcripts do not contain functional HMase, dCTPase, alpha-gt, or kinase mRNA's. However, if E. coli is grown under conditions of limited aeration and then depleted of K+ prior to T4 infection, the T4 RNA which accumulates contains both IE and DE transcripts and functional HMase, dCTPase, and alpha-gt mRNA's. Functional kinase mRNA does not accumulate under these conditions. The results of these experiments indicate that the synthesis of functional DE RNA in the absence of simultaneous protein synthesis, depends on the physiological condition of the cells and the way in which protein synthesis is inhibited. In addition, data is presented which suggests that extensive transcription of DE genes in the absence of protein synthesis results in the inhibition of transcription of certain IE genes.

The influence of potassium and rifampicin on the expression of bacteriophage T2 prereplicative genes in vitro and in vivo

The transcription of specific mRNA from T2 phage DNA by purified Escherichia coli RNA polymerase was markedly affected by the concentration of KCl. Thus the genes for dihydrofolate reductase and deoxynucleotide kinase were transcribed at 50 m m KCl, but at 200 m m KCl the synthesis of the kinase mRNA was nearly eliminated and that of the reductase was increased slightly. Rifampicin-resistant initiation complexes formed at 200 m m KCl failed to produce appreciable kinase mRNA despite reduction of the KCl concentration to 50 m m following initiation of RNA chain elongation. This result suggests that the RNA polymerase holoenzyme does not bind to deoxynucleotide kinase promoters in the presence of 200 m m KCl in a manner capable of initiating synthesis of this message. Initiation complex formation at 50 m m KCl, however, permitted the synthesis of deoxynucleotide kinase mRNA at 200 m m KCl indicating that once formed the initiation complex is capable of producing message even at high salt. The KCl effect on gene expression was reproduced in a coupled E. coli cell-free system directed by T2 DNA. However, in this case the inhibition of protein synthesis at high concentrations of KCl was due primarily to the chloride ions and could be alleviated by substituting acetate for chloride. The synthesis of mRNA in the coupled system was similar with either anion. Analysis of proteins synthesized in the coupled system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, like deoxynucleotide kinase, the synthesis of several additional gene products was influenced by the concentration of KCl. The genes coding for these products, including deoxynucleotide kinase, belong to a class whose transcription is reduced or eliminated in vivo on addition of rifampicin at 1 min postinfection and are referred to as “quasilate.” Evidence is presented to indicate that the mRNAs for deoxynucleotide kinase, dihydrofolate reductase, and deoxycytidylate deaminase are produced in excess of the levels needed for optimal synthesis of these enzymes during normal infection.