Mom Promoter Research Articles

Silencing of toxic gene expression by Fis

Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.

Mutations in β′ subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the β' subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the β' subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the β' subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance.

Conformational Changes Triggered by Mg2+ Mediate Transactivator Function

Transactivator protein C of bacteriophage mu is essential for the transition from middle to late gene expression during the phage life cycle. The unusual, multistep activation of mom promoter (P(mom)) by C protein involves activator-mediated promoter unwinding to recruit RNA polymerase and subsequent enhanced promoter clearance of the enzyme. To achieve this, C binds its site overlapping the -35 region of the mom promoter with a very high affinity, in Mg(2+)-dependent fashion. Mg(2+)-mediated conformational transition in C is necessary for its DNA binding and transactivation. We have determined the residues in C which coordinate Mg(2+), to induce allosteric transition in the protein, required for the specific interaction with DNA. Residues E26 and D40 in the putative metal binding motif (E(26)X(10)D(37)X(2)D(40)) present toward the N-terminus of the protein are found to be important for Mg(2+) ion binding. Mutations in these residues lead to altered Mg(2+)-induced conformation, compromised DNA binding, and reduced levels of transcription activation. Although Mg(2+) is widely used in various DNA transaction reactions, this report provides the first insights on the importance of the metal ion-induced allosteric transitions in regulating transcription factor function.

Dual Role for Transactivator Protein C in Activation of mom Promoter of Bacteriophage Mu

Transactivator C protein of bacteriophage Mu activates the mom gene of the phage by an unusual mechanism. DNA binding by C to its site results in unwinding of the neighboring sequences, realigning the out-of-phase promoter elements to facilitate RNA polymerase (RNAP) binding. High level stimulation of a C-independent constitutive promoter mutant (where RNAP is already bound) by the transactivator suggested an additional mechanism of transcription activation at a step after RNAP recruitment. In this study, we have investigated the various steps of promoter-polymerase interactions during transcription initiation by using both the promoter mutant and a positive control (pc) mutant of C protein. The transactivator does not influence formation of the open complex or its stability after facilitating the RNAP binding. However, at a subsequent step, the protein exerts an important role, enhancing the promoter clearance by increasing the productive RNAP.promoter complex. The pc mutant of the transactivator C is compromised at this step, supporting the additional downstream role for C in mom transcription activation. We suggest that this unusual multistep activation of Pmom has evolved to ensure irreversibility of the switch during the late lytic cycle of the phage.

Identification of the domains for DNA binding and transactivation function of C protein from bacteriophage Mu.

The C protein, a middle gene product of bacteriophage Mu, is the determinant of the transition from middle to late gene expression. C activates transcription from four late gene promoters, P(lys), P(I), P(P), and P(mom) by binding to a site overlapping their -35 elements. Site-specific, high-affinity binding of C to its recognition sequence results in both axial and torsional distortion of DNA at P(mom), which appears to play a role in recruitment of RNA polymerase to the promoter for mom gene transactivation. To identify the regions of C protein important for its function, deletion and site-directed mutagenesis were carried out. We demonstrate here that a helix-turn-helix (HTH) motif located toward the carboxy terminal end of the protein is the DNA-binding domain and amino acid residues involved in transactivation overlap the HTH motif. Mutagenesis studies also aided in the identification of the region important for dimerization. Structure-based sequence alignment and molecular modeling in conjunction with mutational analysis suggest that the HTH motif is part of a three-helix bundle, with remarkable similarity to paired (prd), a developmental regulatory protein from Drosophila. Additional key residues identified in the model to be crucial for C protein structure and DNA binding were shown to be important by mutagenesis. These results provide a structural framework for C function and insight into the mechanism of transactivation at the mom promoter.

Transcriptional activator C protein-mediated unwinding of DNA as a possible mechanism for mom gene activation

The bacteriophage Mu mom gene encodes the unique DNA-modification function of the phage. Regulation of the mom gene at the transcriptional level is brought about by the transactivator protein C of the phage. The mom promoter is an activator-dependent weak promoter having poor −10 and −35 elements separated by a 19 bp suboptimal spacer region. These features could constrain RNA polymerase occupancy at the promoter. Here, we have probed into the mechanism by which C protein acts as a transcriptional activator at P mom . In vivo dimethyl sulfate footprinting studies demonstrate C protein-mediated asymmetric distortion of its specific site at the mom regulatory region. Using a coupled topoisomerase assay, we demonstrate that C protein induces the unwinding of DNA. This C-mediated unwinding seems to be localised to the 3′ flanking region of the C binding site located adjacent to and overlapping the −35 element of P mom . These results suggest that C protein-mediated torsional changes could be reorienting the −10 and −35 elements to a favorable conformation for RNA polymerase occupancy at the mom promoter.

Bidirectional transcription in the mom promoter region of bacteriophage Mu

Transcription of the Mu mom operon requires activation by the phage gene product, C, a site-specific DNA binding protein. Previous in vivo and in vitro footprinting studies showed that Escherichia coli RNA polymerase (Eσ 70=RNAP) bound the wild-type (wt) mom promoter (P mom ) region in the absence of C; this site, now designated momP2 (−11 to −64), is slightly upstream of, but overlapping with, momP1 (+16 to −49), the functional binding site for mom operon (rightward) transcription. The location/distribution of KMnO 4-sensitive sites on the two DNA strands suggested that RNAP bound at momP2 was in an open-complex, but that transcription was in the opposite direction. Here, we used both runoff transcription and reverse transcriptase-primer extension sequencing to provide direct evidence that in the absence of C protein, RNAP carries out leftward transcription from momP2 both in vitro and in vivo. In addition, the 5′ ends of these transcripts were mapped to the same upstream initiation site, −58G, relative to the initiation site of C-activated rightward transcription. We also present evidence that leftward transcription from momP2 requires RNAP recognition of an UP-element by the carboxyl-terminal domain of the α subunit.

Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C.

Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.

Differential binding of RNA polymerase to the wild type Mu mom promoter and its C independent mutant: a theoretical analysis

Using the theoretical model for DNA bending we have analyzed the Mu mom promoter wild type and its mutant tin7 which showed differential binding to the RNA polymerase. We have demonstrated here the structural change as a result of the point mutation which may be responsible for the altered binding of RNA polymerase. Analysis using both sets of parameters essentially gives the same result.

The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter.

Expression of the phage Mu mom gene is transcriptionally regulated by DNA methylation. Three GATC sites upstream of the mom promoter have to be methylated by the Escherichia coli deoxyadenosine methylase (Dam) to allow initiation of transcription. An E. coli dam strain was mutagenized with Tn5 in an attempt to isolate mutants which allow mom gene expression. Three independent Tn5 mutants were isolated, each mapped to a gene at 89.6 min which we designate momR. The wildtype gene was cloned and sequenced, it encodes a protein of 305 amino acids. The protein belongs to a group of related bacterial activators recently identified as the LysR family (Henikoff et al., 1988). MomR protein was overproduced and purified. Expression of momR is autoregulated; MomR binds to a 43 bp region upstream of its coding sequence. In the mom promoter MomR protects a 43 bp region containing the three GATC sites. Specific binding to these sequences was observed only with unmethylated DNA. Fortuitously, we learned that MomR is identical to OxyR, a regulatory protein responding to oxidative stress. We discuss the implications of this control for Mu development.

Role of bacteriophage Mu C protein in activation of the mom gene promoter

The phage Mu C gene product is a specific activator of Mu late gene transcription, including activation of the mom operon. Fusion of the C gene to the efficient translation initiation region of the Escherichia coli atpE gene allowed significant overproduction of C protein, which was subsequently purified and assayed for DNA binding by gel retardation and nuclease footprinting techniques. C protein binds to a site immediately upstream of the -35 region both of the mom promoter and the related phage D108 mod promoter. The location of the mom promoter has been determined by primer extension. Upstream deletions extending more than 3 base pairs into the C-binding site abolished activation of the mom promoter in vivo. In vitro binding of C was not significantly affected by DNA methylation. A second, C-dependent promoter was identified just downstream of the C coding region; comparison with the mom promoter revealed common structural elements.

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence

Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein. Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter. S1 nuclease protection experiments were used to map RNA 5' ends in the three new regions. Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome. Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene. A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli sigma 70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E. coli -35 consensus. We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity. Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent beta-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation. As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage. The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.

The mom gene of bacteriophage Mu: the mechanism of methylation-dependent expression.

Transcription of the DNA modification gene (mom) of bacteriophage Mu requires methylation of three GATC sites upstream of the mom promoter by the Escherichia coli deoxyadenosine methylation function (Dam). The three sites map within a 40-bp segment termed region I. Small deletions, inversions, duplications and specific point mutations have been introduced in region I. Their effect on mom expression has been studied in dam+ and dam strains. Dam-dependent expression of the mom gene requires a specific arrangement of the three GATC sites and the presence of the methylated base in at least two of the three sites. We show that mom specific modification is regulated by a host protein. The Mom function is expressed in dam strains if they are defective in one component of the methylation-instructed mismatch correction system, mutH. We suggest that the product of mutH functions as a transcriptional repressor by binding to region I.

The sequence and mom-transactivation function of the C gene of bacteriophage Mu

The mom gene of bacteriophage Mu encodes a DNA modification function. The gene is regulated on the transcriptional level by Dam-specific methylation and a trans-acting Mu function, and on a post-transcriptional level by the product of gene com. The gene encoding the transactivator has been cloned and mapped. By complementation analysis the activation function (also designated Dad) was shown to be the product of gene C. Transactivation of the mom promoter was shown in the following assay: the mom promoter and N-terminal part of com were fused in frame to lacZ. Cells containing such fusion plasmids were infected with M13 clones expressing C in the presence of IPTG and XGal. Successful transactivation results in the formation of blue plaques. Moreover, we have determined the sequence of gene C and found that it has a coding capacity of 140 amino acids. The promoter for C ( p C ) is likely to be located at least 0.5 kb upstream from the gene. A transcription terminator is found directly downstream from the C-coding region.

The mom gene of bacteriophage Mu: a unique regulatory scheme to control a lethal function

The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5'-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene. Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters. The com gene product itself is not lethal to the cell. The region encoding mom has been cloned in p L expression vectors. The mom gene product, a peptide of 27 kDal, has been visualized on gels. Efficient expression of Mom from p L requires gene com. A fusion between MS-2 polymerase and com has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit. We show that the defect associated with com deletions can be complemented in trans, and propose that com acts as a positive regulator on a post-transcriptional level.

Analysis of the methylation-regulated Mu mom transcript.

The Mu mom gene is transcribed only when DNA is methylated by Dam; this methylation dependence is determined by a region upstream of the gene containing three Dam-methylation sites. The mom operon contains two open reading frames, one of which codes for Mom. Experiments were done to find a function for the other reading frame; it is shown that it does not affect phage immunity or the regulation of mom expression. Deletion mapping and S1 nuclease mapping experiments identify the +1 of the mom mRNA: it is 45 bp downstream of the three Dam-methylation sites. Possible models for the short-distance transcription regulation by methylation are discussed. The mom promoter is surrounded by sequences that are shown to be homologous to the oriC region of E. coli. This suggests a relation between replication and methylation-dependent mom expression. It is shown however, that replication is not essential for mom expression.

Methylation dependent expression of the mom gene of bacteriophage Mu: deletions downstream from the methylation sites affect expression.

The expression of the DNA modification gene (mom) of bacteriophage Mu requires the cellular deoxyadenosine methylase (dam) and a transactivation factor from the phage. By hypothesis, the transcription of mom is activated by methylation of three GATC sequences upstream from the mom gene. We have introduced small deletions at a fourth GATC site located about 140 base pairs downstream from the primary methylation region. Some of the deletions severely affect the mom gene expression. We propose from this analysis that (1) some important elements, possibly the promoter, concerned with the expression of mom are located between nucleotides 840 and 880 from the right end of Mu and (2) the mom protein starts with the codon GTG located at position 810. We favor the hypothesis that methylation turns off transcription upstream, thereby allowing the main mom promoter to function.

Transcription initiation of Mu mom depends on methylation of the promoter region and a phage-coded transactivator.

The product of the bacteriophage Mu gene mom modifies adenine residues of DNA within the consensus sequence CGAGCNPy, providing protection against various restriction endonucleases (ref. 1 and D. Kamp, personal communication cited in ref. 2). The mom gene is only expressed during lytic development of the phage. It is known that mom is nonfunctional in Escherichia coli host mutants in a gene (dam) which itself encodes an adenine methylation system. We show here that the E. coli dam gene is essential for transcription initiation of the mom gene, and that this dependence on dam seems to lie in a short segment preceding the mom coding region, which also contains the mom promoter. The sequence of this segment reveals the presence of dam methylation sites (GATC), and suggests a model for the regulation of mom gene expression based on DNA secondary structure, which may explain why mom is only expressed during phage lytic development. We also show that expression of phage-coded proteins (A, B and C) is needed for transactivation of mom transcription.