P2 Late Promoters Research Articles

Identification of upstream sequences essential for activation of a bacteriophage P2 late promoter.

We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, P(F), which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the -35 region of the promoter. Introduction of a sigma(70) -35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor sigma(70) contacts and helps to recruit RNA polymerase holoenzyme.

Activation of P2 late transcription by P2 ogr protein requires a discrete contact site on the C terminus of the α subunit of Escherichia coli RNA polymerase

Bacteriophage P2 late transcription requires the product of the P2 ogr gene. Ogr-dependent transcription from P2 late promoters is blocked by certain point mutations affecting the α subunits of the host RNA polymerase. An alanine scan spanning the putative activation target in the α C-terminal domain (αCTD) was carried out to identify individual residues essential for Ogr-dependent transcription from P2 late promoters. In addition, the effects of alanine substitutions in the regions of the αCTD previously reported to affect CAP-dependent activation of the lac promoter and UP-element DNA binding were examined. Residues E286, V287, L289 and L290 in helix 3, and residue L300 at the beginning of helix 4, define a surface-exposed patch on the αCTD important for Ogr-dependent activation. These residues, adjacent to the recently identified DNA-binding determinants, constitute a newly identified activation surface for protein:protein contact. Alanine substitutions at some of the residues that affect UP-element DNA binding also impaired activation. This suggests that upstream DNA-α contacts, in addition to α-Ogr contacts, may be important in P2 late transcription. Other residues implicated in the interaction of α with CAP are not required for activation by Ogr, consistent with previous genetic evidence suggesting that these activators contact different sites on the αCTD.

Metal- and DNA-binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: a prokaryotic C4 zinc-finger protein.

Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which contain a Cys-X2-Cys-X22-Cys-X4-Cys presumptive zinc-finger motif. The molecular characterization of these proteins has been hampered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium-containing derivative (CdB). Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS-Cd(II) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification (delta epsilon 248, 24 mM-1 cm-1) indicated the presence of a Cd(Cys-S)4 center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (KD, app 3-4 microM) and the protein was shown to activate transcription in vitro from a promoter-reporter plasmid construct. The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between-70 and -43 relative to the transcription start site, coincident with the consensus sequence, GTTGT-N8-TNANCCA, from -66 to -47 of the 186 and P2 late promoters. Inactive B point mutants were obtained in the putative DNA-binding loop of the N-terminal zinc-finger motif and in a central region thought to interact with the Escherichia coli RNA polymerase alpha-subunit. A truncated B mutant comprising the first 53 amino acids (B1-53) exhibited close to wild-type activity, showed a DNA-binding affinity similar to that of the full-length protein, and could be reconstituted with either Cd or Zn. Gel permeation analysis revealed that B1-53 was a majority dimeric species whereas wild-type B showed larger oligomers. 186 B therefore exhibits a potentially linear organization of functional regions comprising an N-terminal C4 zinc-finger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase.

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered.

Mutational analysis of a bacteriophage P4 late promoter

Transcription from the late Psid promoter of satellite bacteriophage P4 is dependent on the bacterial RNA polymerase carrying the sigma 70 subunit and is positively regulated by the product of the P4 delta gene or the ogr gene of helper bacteriophage P2. Through deletion and mutational analyses of the Psid promoter, we identified mutations in the -10 region and in a region of hyphenated dyad symmetry centered around position -55 that inactivate Psid. Most of these mutations alter base pairs that are highly conserved in the five other delta-activated P4 and P2 late promoters. We propose that the P4 delta and P2 ogr gene products bind the -55 region of the P4 and P2 late promoters.

Deletion analysis of a bacteriophage P2 late promoter

We have fused the promoter ( P F) for the P2 late FETUD operon to the gene ( cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, δ. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned P F is severely impaired in a host strain carrying the rpoA109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of P F were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides −69 and −64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.

Control of gene expression in the P2-related temperate coliphages: IV. Concerning the late control gene and control of its transcription

In this paper we have sequenced four amber mutants and thereby confirmed the gene D (CP65) and gene B (CP67) assignments made in the accompanying paper (Kalionis et al., 1986). We have also studied, by gel electrophoresis, the transcription patterns of gene B in vivo. In a lysogen, gene B is present on a short transcript under autogenous (negative) control. Upon prophage induction, this transcript is amplified, but later in the cycle gene B is present on a larger transcript that originates in the late region. We have detected two copies of an inverted repeat in the promoter region of the B gene that we predict is recognized by the B protein. One arm of this repeat is associated with three of four P2 late promoters, downstream from the start point of transcription. The repeat is not present in the promoter region of P2 ogr. We describe the considerable homology in amino acid sequence seen with the late control proteins 186 gpB, P4 gpδ and P2 gpOgr, and present a working model for control of late gene transcription.

Bacteriophage P2 late promoters: II. Comparison of the four late promoter sequences

The late genes of bacteriophage P2 are clustered into four transcription units. We have reported the transcription initiation sites for two of the late messenger RNAs, encoding genes QP and ONMLKRS. We have now located the 5′ ends of the two remaining late mRNAs. The first gene in the VJHG transcription unit has been located by DNA sequence determination of the single nucleotide change in a V amber mutant. Location of the first gene in the FETUD transcription unit has been inferred from the DNA sequence. The 5′ ends of the mRNAs for these two transcription units were located by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S 1. Similar protection of hybrids using RNA that had been 5′ end-labeled with [ α- 32P]GTP and guanylyl transferase confirmed that these 5′ termini resulted from initiation of transcription. The DNA sequences preceding the P2 late transcription starts are different from the Escherichia coli promoter consensus sequences at −10 and −35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The four P2 late promoters do share sequence homologies in the −10 and −35 regions, however, and several additional homologies further upstream. P2 late gene expression also appears to involve negative regulation by a product of the ONMLKRS gene cluster. When cells are infected with P2 polar O amber mutants, a marked increase in the levels of proteins encoded by the other three gene clusters is observed. This increase is reflected in the amounts of late mRNAs, suggesting that RNA synthesis is normally repressed or that late mRNAs are more labile in the presence of a gene product from the ONMLKRS transcription unit. Satellite phage P4 induces P2 late gene expression without the usual requirement for P2 DNA replication. The 5′ ends of the P2 late mRNAs are the same during P4 transactivation as during normal P2 late gene expression. Thus, the regulation of P2 late gene expression by P4 does not involve altered promoter selection.

Bacteriophage P2 late promoters: Transcription initiation sites for two late mRNAs

Divergent transcription of two of the bacteriophage P2 late mRNAs, encoding genes QP and ONMLKRS, is initiated from opposite strands of the DNA in a region near the left end of the P2 genome. The first gene in each of these transcription units (P and O) has been located in the nucleotide sequence by amino-terminal sequence analysis of the P gene product and by DNA sequence determination of the single nucleotide changes in two O amber mutants. The 5' ends of the P and O gene mRNAs are separated by 109 nucleotide pairs in the DNA template. The locations of these 5' termini were determined by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S1. Sequence analysis of mRNA that had been labeled at the 5' end with [alpha-32P]GTP and guanylyl transferase confirmed that these termini resulted from initiation of transcription. The DNA sequences preceding the O and P transcription starts have poor homologies to the bacterial promoter consensus sequences at -10 and -35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The O and P promoter regions also have no detectable homology to each other in the -10 or -35 regions, and are unusually G + C-rich. There are, however, blocks of sequence homology within the transcribed region of each of these two late operons near the 5' end. Satellite phage P4 induces P2 late gene expression without the usual requirement for P2 DNA replication. The 5' ends of the P2 P and O gene transcripts are the same during P4 "transactivation" as during normal P2 late gene expression. Thus the regulation of P2 late gene expression by P4 does not involve a change in the site for initiation of transcription.