Early Operon Research Articles

An unusual promoter controls cell‐cycle regulation and dependence on DNA replication of the Caulobacter fliLM early flagellar operon

Transcription of flagellar genes in Caulobacter crecentus is programmed to occur during the predivisional stage of the cell cycle. The mechanism of activation of Class II flagellar genes, the highest identified genes in the Caulobacter flagellar hierarchy, is unknown. As a step toward understanding this process, we have defined cis-acting sequences necessary for expression of a Class II flagellar operon, fliLM. Deletion analysis indicated that a 55 bp DNA fragment was sufficient for normal, temporally regulated promoter activity. Transcription from this promoter-containing fragment was severely reduced when chromosomal DNA replication was inhibited. Extensive mutational analysis of the promoter region from -42 to -5 identified functionally important nucleotides at -36 and -35, between -29 and -22, and at -12, which correlates well with sequences conserved between fliLM and the analogous regions of two other Class II flagellar operons. The promoter sequence does not resemble that recognized by any known bacterial sigma factor. Models for regulation of Caulobacter early flagellar promoters are discussed in which RNA polymerase containing a novel sigma subunit interacts with an activation factor bound to the central region of the promoter.

Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda.

The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons. The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E. coli factors NusA, NusB, NusG, and ribosomal protein S10. We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR. NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction. At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites. In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites. This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome.

Integration host factor activates the Ner-repressed early promoter of transposable Mu-like phage D108.

The lytic-lysogenic switch in transposable, Mu-like bacteriophage D108 is governed by two divergent and slightly overlapping transcription units originating from the Pe and Pc promoters. DNase I footprinting and in vivo mutational analysis suggest that lysogeny is maintained by c-repressor occupancy of the O2 operator, which precludes RNA polymerase from binding to Pe. Lytic development is controlled by the Ner repressor, which binds to a site symmetrically situated between the converging promoters and, in the absence of other factors, prevents RNA polymerase from binding to either Pc or Pe. DNase I protection and potassium permanganate hypersensitivity in the presence of integration host factor (IHF), which binds and alters the DNA structure upstream of Pe, revealed that RNA polymerase was able to bind Pe irrespective of the Ner.DNA-bound complex, and partially unwind the Pe "-10 region." Ner repression of Pe transcription in vitro was significantly more effective in the absence of IHF. Using a cloned D108 early region-lacZ fusion in IHF-deficient and -proficient backgrounds, we also demonstrate this host factor's affect on ner-repressed Pe in vivo, and generate a system for isolating mutants in the regulatory genes and sites controlling this genetic switch. D108 lytic growth is proposed to occur through IHF-mediated activation of the phage Ner-repressed early operon.

Control of gene expression in the temperate coliphage 186: VIII. Control of lysis and lysogeny by a transcriptional switch involving face-to-face promoters

The lysogenic and early lytic operons of the temperate coliphage 186 are transcribed divergently. Primer extension mapping of the 5' ends of these in vivo transcripts showed that the rightward lytic promoter, pR, and the leftward lysogenic promoter, pL, are arranged face-to-face, with their transcripts overlapping by 60 bases. We examined the control of transcription from pR and pL using galK as a reporter gene. The product of the lysogenic cI gene strongly repressed pR transcription while allowing pL transcription. The product of the lytic apl gene (formerly CP75) strongly repressed pL transcription while allowing pR transcription. Thus, the cI-pR-pL-apl region functioned as a transcriptional switch, determining whether transcription was lytic or lysogenic. Also, the cI gene product was able to stimulate pL, possibly by alleviating an inhibition of pL transcription caused by convergent transcription from pR. Other consequences of the face-to-face promoter arrangement are discussed.

Characterization of the C operon transcript of bacteriophage Mu

Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.

Purification and characterization of the Ner repressor of bacteriophage Mu

The Ner protein of bacteriophage Mu acts as a λ cro-like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5′-ANPyTAPuCTAAGT-3′, separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar λ cro protein, gel filtration experiments show the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.

Effects of all single base substitutions in the loop of boxB on antitermination of transcription by bacteriophage λ's N protein

The 'N' antitermination proteins of lambdoid bacteriophages are essential for overcoming multiple transcription terminators located within the major early operons of these phages (1). In order for N proteins to function, a genome sequence specifying N utilization, nut, must be located within an operon, between the promoter and the terminators (2). Two components have been identified within nut: 8-base boxA, conserved among different phages and implicated in the recognition of host NusA protein, required for N function (3); 15-base boxB, an interrupted palindrome (4), diverged in sequence among different lambdoid phages and hypothesized to be the site of recognition for different N proteins, also diverged in sequence (5). Here we apply a plasmid for testing termination and antitermination of transcription (6) to identify mutations at all positions in the 5-7 base loop of lambda's boxB. Almost every base change at any position within the 5-7 base boxB loop was found to constrain antitermination of transcription by the N protein of bacteriophage lambda. These observations extend previous mutational knowledge of nut (7) and are consistant with the hypothesis that the boxB loop is the direct site of recognition for N protein. Variations among the effects of different base changes suggest differential contacts between N protein and bases of the boxB loop, whether in DNA or RNA.

Sequences at the right end of Escherichia coli phage Mu DNA influence expression of the early operon

The early operon of Mu DNA was fused transcriptionally to the galK indicator gene using the HpaI site at the end of the B gene. From this plasmid the indicator gene was expressed at low and high temperature indicating that the repressor gene is not expressed. When this construction was complemented in cis with the right end 115-bp sequence of Mu DNA, repression is restored at low temperature. It could be shown that the 115-bp sequence acts in both orientations and must be present in cis. Deletion of attL, at the right end of Mu DNA, does not interfere with the effect exerted by the 115-bp sequence.

Sequences at the right end ofEscherichia coliphage Mu DNA influence expression of the early operon

The early operon of Mu DNA was fused transcriptionally to the galK indicator gene using the HpaI site at the end of the B gene. From this plasmid the indicator gene was expressed at low and high temperature indicating that the repressor gene is not expressed. When this construction was complemented in cis with the right end 115-bp sequence of Mu DNA, repression is restored at low temperature. It could be shown that the 115-bp sequence acts in both orientations and must be present in cis. Deletion of attL, at the right end of Mu DNA, does not interfere with the effect exerted by the 115-bp sequence.

The cox protein of bacteriophage P2 inhibits the formation of the repressor protein and autoregulates the early operon.

The cox gene is the first gene of the early operon of bacteriophage P2. The early promoter Pe and the repressor promoter Pc are located close to each other and in such a way that their transcripts have opposite polarity and show an overlap of about 30 nucleotides. The expression of the early operon and of the C gene was studied in vivo by using fusions to a promoterless cat (chloramphenicol acetyl transferase) gene. The results show that the Cox protein negatively autoregulates the early operon and inhibits the formation of the repressor C. By measuring the efficiency of plating of a series of P2 virulent deletion mutants on bacteria carrying a cloned cox gene, the site of action of the Cox protein was mapped within the Pe-Pc region. The stimulatory effect of the C protein on expression of the Pc promoter was found to be due to inhibition of transcription from Pc; this was demonstrated by mutating Pe which showed that loss of transcription from Pe stimulated transcription from Pc. Hence, this is a case of regulation of gene expression by convergent transcription. By cloning the region C-Pe-Pc-cox such that the cat and kan genes are expressed from Pc and Pe, respectively, it was shown that only one of the resistances (Cm and Km) was expressed. This mimics the choice between lysogeny and lytic growth of the phage. The 'lysogenic' state was very stable whereas the 'lytic' state flipped to the 'lysogenic' at a somewhat higher frequency. The presence of a cloned cox gene drastically stimulated the formation of free phage from a P2-lysogen and dramatically reduced the frequency of lysogenization after P2 infection. We conclude that the pleiotropic effects of the cox (control of excision) gene, namely effects on lysogenization, formation of free phage and site-specific P2 recombination, can be explained by the effect of the Cox protein on the activity of the promoters Pc and Pe.

DNA sequences of bacteriophage P2 early genes cox and B and their regulatory sites.

Part of the early operon of the temperate phage P2 of Escherichia coli, including genes cox (involved in prophage excision) and B (required for phage specific DNA synthesis), was sequenced. The results are consistent with an early promoter spanning the repressor binding sites, a leader sequence of about 80 bases which overlaps the leader sequence of the repressor gene for about 30 bases, and coordinate transcription of genes cox and B with a termination signal after the B gene. In addition, the data provide amino acid sequences for the Cox and B proteins of 91 and 166 residues, respectively and reveal a hitherto undetected coding sequence between genes cox and B that has the potential to produce a very basic polypeptide of 56 residues. Slight structural similarities between the P2 Cox protein and the analogous Xis protein of phage lambda were noted and the P2 B gene product was compared with proteins that interact with the DnaB protein of E. coli.

Conservation of genome form but not sequence in the transcription antitermination determinants of bacteriophages λ, φ21 and P22

Comparisons are made among DNA sequences upstream from terminators in both leftwards and rightwards early operons of related coliphages λ, φ21 and P22. These sequences include both left and right determinants of response to phage-coded antitermination proteins, “ N”, as well as the N structural genes themselves. Despite almost total disparity of DNA sequence, the three genomes can be discerned to include the same elements in the same order and spacing: downstream from the early left promoter are sequentially a site of recognition for host nusA protein, a dyad symmetry “ nut” essential for N function in λ, overlapping sites for processing of the transcript by RNAase III and then the N structural genes; downstream from the cro gene on the right are sites of nusA recognition and nut dyad symmetries homologous to those on the left. Because the N proteins of λ, φ21 and P22 do not for the most part complement each other, a specific site of N recognition has been postulated for each N-responding operon. The nut dyad symmetry qualifies as such a site, since the loop of the left dyad in λ is marked by mutations that block N function leftwards, and since DNA sequences here show close homology between the loops of left and right dyads for each phage, but less if not little homology for different phages.

Sequence analysis of a region from the early right operon in phage P22 including the replication genes 18 and 12

A comparison of a 3000-bp sequence of Salmonella phage P22, coding for gene c1 and the replication genes 18 and 12, with the analogous cII- O- P region of coliphage λ permits the localization of transcriptional signals, an oop RNA species, and the origin of replication ( ori) within gene 18. Gene c1 and the amino terminal region of gene 18 show homology to the respective λ genes. In the ori domain of the replicator proteins the homology to ø82 is most pronounced. Of two λ: rep P22 hybrids (λ with replication genes of P22) analysed, one codes for a hybrid 0/18 protein with 30 N-terminal amino acids coded for by λ. Gene 12 is nonhomologous to its λ counterpart (gene P), but closely related to dnaB of Escherichia coli. A ren gene is missing, whereas two open reading frames (ORFs) distal to gene 12 are almost identical to those in the λ nin region. We try to account for the occurrence and location of highly conserved sequences among the lambdoid phages by assigning them a role in recombinational reassortment of functional units during the evolution of this phage family.

Physical evidence for early transcription in intracellular bacteriophage P2 DNA

Escherichia coli were infected with bacteriophage P2, and the phage DNA replication was blocked by a mutation in the phage A gene. The resulting intracellular DNA molecules contained displacement loops, which were shown to contain RNA. The positions of these R loops were mapped by electron microscope partial denaturation mapping, and 90% were found to be located in the same region on the DNA, spanning less than 15% of the P2 chromosome. Also, most of the R loops have a common left end located at 77 ± 1.4% on the P2 DNA map, which corresponds well with the position of the promoter for the P2 early genes as determined by the position of several P2 virulent deletion mutations. We conclude that the observed R loops on intracellular P2 DNA represent transcription from the promoter for the early operon in P2.

Kinetics of P22 early gene expression suggests a cro-like regulatory function.

Analysis of phage-specified protein synthesis after phage infection of UV-irradiated cells shows a turn-off of early gene expression, a regulatory event that is independent of the known P22 regulatory functions. This supports the suggestion of a cro lambda-like function in P22. We have identified the products of genes 18 and int as contributing to the complex 40,000 dalton band in our SDS-polyacrylamide gels. Both gene products appear to be subject to regulation by the cro-like function of P22. Proteins of 33,000, 29,000, 27,000, 25,000, and 24,000 MW, specified by as yet unidentified P22 genes of the early leftward operon, are regulated by the same function. Our data suggest that the cro-like function is expressed from the early rightward operon.

New genes and promoters suggested by the DNA sequence near the end of the coliphage T7 early operon

We have employed the dideoxynucleotide chain-terminating method to determine the nucleotide sequence of T7 DNA between the physical map positions 18.9% and 19.8%. The most striking features of this sequence are two perfect 21-basepair repeats, each of which appears to contain a promoter for late transcription. In each case the promoter sequence incorporates a putative translational terminator on its left (5'-side of the "sense" strand), and overlaps a potential ribosome-binding site on its right. The region probably lies immediately distal to the early operon, and may contain two short, hitherto unreported protein-coding sequences.

Mutational analysis of the operators of bacteriophage lambda

Oc mutations in the operators of bacteriophage lambda have been used to analyze the functional organization of the operators. In each operator, repressor binding sites 1 and 2, as identified biochemically, were found to be primarily responsible for the repressor affinity of the operators in vitro and for the repression of lytic functions in vivo. In addition, both sites were shown to be involved in the action of cro product at the operators. The data obtained have been used to estimate the repressor affinities of the individual binding sites. These affinities suggest that repressor bound at OR1 and OR2 interacts cooperatively. The results obtained support a model for repression of the early lambda operons where repressor bound at binding sites 1 and 2 interferes with RNA polymerase binding to the promotor sites.

High resolution electron microscopic studies of genetic regulation

High magnification electron microscopic methods were used to study DNA fragments and regulatory proteins binding to DNA fragments containing the lambda phage early rightward operon and the lac operon. It was found that DNA lengths and repressor or RNA polymerase binding positions could be determined with a precision of about ± five base-pairs. DNA and protein were positively stained with uranyl formate. This staining yeilds reproducible differences in the shapes of bound proteins, but shows no reproducible internal structure. RNA polymerase alone was able to bind to the lambda promoter pr, but the presence of catabolic gene activator protein was required for detectable RNA polymerase binding to the lac promoter. Less than a 2% shortening was observed in the measured length of a 203 base-pair lac DNA fragment upon the binding of repressor to operator and the DNA showed only a slightly increased tendency to bend at the site of repressor or RNA polymerase binding. Similar microscopic studies should be possible for other systems in which proteins bind to DNA.

Stimulation of RNA chain initiation by the N gene protein of bacteriophage lambda.

RNA transcription from the early leftward operon of the bacteriophage λ was studied. Hybridization to the separated DNA strands of the wild type phage, and to a biotin transducing phage containing viral sequences of only the extreme right or promoter proximal segment of this operon, allowed measurement of the rate of RNA chain initiation with respect to the total amount of RNA transcribed from this operon. Procedures which reduced either the amount of N gene protein synthesized, or its activity, were seen to depress transcription from the promoter proximal region approximately as much as from the entire operon. The RNA made when transcription was depressed did not have a conspiciously lower molecular weight, and the DNA-polymerase-RNA complex contained a correspondingly reduced number of polymerase molecules. All these observations are consistent with, and suggest, that under these conditions the function of the protein specified by gene N is to increase the frequency of initiation of RNA chains.

Constitutive expression of bacteriophage P2 early genes resulting from a tandem duplication.

A tandem-duplication mutant of bacteriophage P2 was isolated. Physically, its particles are characterized by a higher buoyant density and lower heat stability than the wild type, both consequences of increased DNA content. Genetically, the mutant is easily recognized by its insensitivity to control by the immunity-specific repressor. The duplication covers part of gene B, necessary for phage DNA replication. To explain the immunity-insensitivity of the duplication it is proposed that the promoter, but not the operator site, in the early gene operon is duplicated in this mutant. By crosses with a gene-B mutant, a recombinant carrying a heterozygous duplication was isolated.