RESUMEN

Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions. Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases. The gpNu1 subunit was obtained from E. coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein. Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively. The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes. Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends. The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments. This information is key to design new plasmids to overproduce these domains for further structural analysis.

Asunto(s)

RESUMEN

Gene product W (gpW), the head-tail joining protein from bacteriophage lambda, provides a fascinating model for studying protein interactions. Composed of only 68 residues, it must interact with at least two other proteins in the phage, and probably with DNA. To study the structural and functional properties of gpW, plasmids were constructed expressing gpW with hexahistidine tag sequences at either the N or C terminus. The purified wild type fusion proteins were found to be stably folded and biologically active. The protein is monomeric as judged by equilibrium ultracentrifugation, and appears to unfold by a cooperative two-state mechanism. Circular dichroism studies indicate that the protein is 47% helical, with a T(m) of 71.3 degrees C, and a DeltaG(u) of 3.01 kcal/mol at 25 degrees C. Mutagenesis of the three hydrophobic C-terminal residues of gpW showed that they are critical for activity, even though they do not contribute to the thermodynamic stability of the protein. Using secondary structure prediction as a guide, we also designed destabilized gpW mutants. The hydrophobic nature of the gpW C terminus caused these mutants to be degraded by the ClpP-containing proteases in Escherichia coli.

Asunto(s)

RESUMEN

DNA maturation in bacteriophage lambda is the process by which the concatemeric precursor DNA is cleaved at sites called cos to generate mature lambda DNA molecules. These DNA molecules are then packaged into procapsids, the empty capsid precursors. The enzyme that catalyses these events is lambda DNA terminase. It is composed of two subunits, made of 181 and 641 amino acids, the products of genes Nu1 and A, respectively. The product of the FI gene (gpFI) stimulates the formation of an intermediate in capsid assembly called complex II, which contains a procapsid, terminase and DNA. The mechanism of stimulation remains unknown. It has been suggested that gpFI may also stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. Mutants in FI fail to mature and package DNA but, in comparison with other capsid gene mutants, FI mutants are leaky. Second site mutants of FI phages, called 'fin' (for FI independence), bypass the necessity for gpFI. These mutants were originally localized to the region of Nu1 and A and are of two classes: finA includes those that induce the synthesis of fourfold more gene A product (gpA) than wild-type phages, and finB includes those that produce normal amounts of gpA. Whereas all finA mutants analysed map to Nu1, finB mutants have been found both in E and in Nu1. The existence of E mutants able to bypass the necessity for gpFI in vivo shows that gpE and gpFI interact, directly or indirectly. Here we have analysed and sequenced two finA mutants and one finB mutant. All of these map in Nu1. Of the two finA mutants, one corresponds to an Ala163Ser change and the other is a silent mutation. It is likely that the finA mutations alter mRNA conformation in a manner that results in an increase in the efficiency of A mRNA translation. The fourfold increase in gpA synthesis translates into a 10-fold increase in terminase activity. These results show that terminase overproduction is sufficient to bypass the necessity for gpFI and that such an overproduction can be achieved by changes in the efficiency of translation of A due to subtle changes in the sequence upstream of the gene. The finBcs103 mutation is a His-87-->Tyr change in Nu1. Therefore, an alternative way in which to bypass the requirement for gpFI involves an alteration in the structure of gpNu1. It is likely that the altered gpNu1 would increase cleavage and packaging efficiency directly or indirectly. We have determined that DNA cleavage in vivo does not occur in the absence of gpFI. Therefore it seems that gpFI somehow facilitates an otherwise latent capacity of terminase to autoactivate its nucleolytic activity.

Asunto(s)

RESUMEN

The functions of most of the 10 genes involved in phage lambda capsid morphogenesis are well understood. The function of the FI gene is one of the exceptions. Mutants in FI fail to mature and package DNA. The gene product (gpFI) seems to act as a catalyst for the formation of an intermediate in capsid assembly called complex II, which contains a procapsid (an empty capsid precursor), terminase (the enzyme that cleaves the DNA precursor and packages it into the procapsid) and DNA. The mechanism for this stimulation remains unknown. It has also been reported that gpFI appeared to stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. In comparison with other head-gene mutants, FI mutants are leaky, producing approx. 0.1 phage per infected cell. Some second-site revertants of FI- phages, called 'fin', that bypass the necessity for gpFI, have been isolated and found to harbour a mutation in the genes that code for the two subunits of terminase. In the course of mapping additional fin mutants, it was discovered that some mapped outside the terminase genes. To localize the mutations, restriction fragments of fin mutant DNAs were subcloned into plasmids and their ability to contribute to fin function was determined by marker-rescue analysis. The location of the fin mutation was further delineated by deletion analysis of a plasmid that was positive for fin. This showed that some fin mutations mapped to a region comprising genes E, D and a portion of C. The sequencing of this entire region in several fin isolates showed that the fin mutations are clustered in a small region of gene E corresponding to a portion of 26 amino acid residues of the coat protein (gpE). We have called this region of the protein the EFI domain. All the mutations result in an increase in positive charge relative to the wild-type protein. These results suggest that DNA maturation and packaging are in part controlled by an interaction between gpFI and capsid gpE.

Asunto(s)

RESUMEN

The three-dimensional structures of the procapsid and of the mature capsid of bacteriophage lambda were determined to a resolution of approximately 3.4 nm by cryo-electron microscopy and image processing. The mature lambda capsid contains two major proteins, gpE and gpD, arranged on a T = 7 lattice, with gpE arranged as hexamers and pentamers and gpD arranged as trimers. The hexamers and pentamers in the virion display a cartwheel-like structure, with skewed spokes (or arms) radiating out from a central hexameric hub. The thimble-shaped gpD trimers are superimposed on the trivalent interaction point of these arms. A reconstruction of a lambda D- mutant capsid to lower resolution shows no trace of these trimers, thus revealing the interactions of the underlying arms. The procapsid has elongated, irregularly shaped hexamers with gpE subunits set perpendicularly to the capsid surface.

Asunto(s)

RESUMEN

The Nu3 gene of bacteriophage lambda resides within a cluster of genes that specify structural components of the bacteriophage head. Previous experiments indicate that the Nu3 gene product (gpNu3) is associated with immature proheads but is not detectable in mature proheads or bacteriophage particles, hence its classification as a scaffolding protein. The Nu3 gene has been cloned and overexpressed, and its protein product has been purified. The purified protein is biologically active, as demonstrated by its ability to complement a gpNu3-deficient extract in an in vitro assembly reaction. The sequence of the amino terminus of the protein indicates that translation of Nu3 starts at nucleotide position 5,342 on the standard lambda DNA sequence, yielding a protein with a calculated Mr of 13,396. A combination of gel exclusion chromatography and velocity sedimentation gradient data indicates that gpNu3 possesses an unusually elongated shape.

Asunto(s)

RESUMEN

To study the specificity and identity of NF-lambda 2, a ubiquitous murine nuclear factor that interacts specifically with the promoter of the lambda 2-chain gene and stimulates its transcription, competition experiments were carried out using DNA fragments from various immunoglobulin regulatory elements. The results showed that a fragment containing the H-chain enhancer competed efficiently for the binding of NF-lambda 2. Dissection of the H-chain enhancer revealed that the microE3 motif contributed the competing ability. Additionally, a regulatory region found in the adenovirus major late promoter, which interacts with the human general transcription factor USF, competed very efficiently for binding to NF-lambda 2. This region contains a sequence, CACGTGAC, which is identical to a region within the NF-lambda 2 motif. The pattern of complexes formation using oligonucleotide probes corresponding to the NF-lambda 2 and USF motifs were identical, and they both differed from that displayed by the E3 probes. Antisera against different domains of USF also react specifically with NF-lambda 2 showing that this factor is antigenically related, if not identical, to USF. Furthermore, the activity of the lambda 2 promoter in an in vitro transcription assay was significantly reduced when the nuclear extract used was USF-depleted. Addition of exogenous USF to this extract restored the transcription activity. Therefore, we conclude that NF-lambda 2 is the murine homologue of USF.

Asunto(s)

RESUMEN

The terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda DNA and its packaging into phage proheads; it is composed of the products of the lambda Nul and A genes. We have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill recA strains of Escherichia coli. Sixty-three different spontaneous mutations and 13 linker insertion mutations were isolated by this method and analyzed. Extracts of cells transformed by mutant plasmids displayed variable degrees of reduction in the activity of one or both terminase subunits as assayed by in vitro lambda DNA packaging. A method of genetically mapping plasmid-borne mutations in the A gene by measuring their ability to rescue various lambda Aam phages showed that the A mutations were fairly evenly distributed across the gene. Mutant A genes were also subcloned into overproducing plasmid constructs, and it was determined that more than half of them directed the synthesis of normal amounts of full-length A protein. Three of the A gene mutants displayed dramatically reduced in vitro packaging activity only when immature (uncut) lambda DNA was used as the substrate; therefore, these mutations may lie in the endonuclease domain of terminase. Interestingly, the putative endonuclease mutations mapped in two distinct locations in the A gene separated by a least 400 bp.

Asunto(s)

Asunto(s)

RESUMEN

Proheads of bacteriophage lambda which carry the connector of phage phi 29 instead of that of lambda have been produced in vitro. These hybrid proheads have a structure similar to that of normal lambda proheads. Furthermore, the chimeric proheads can package both lambda and phi 29 DNA. These data show that the connector domains involved in both head assembly and DNA packaging are functionally similar. The DNA-containing lambda-phi 29 proheads can be complemented in vitro with phi 29 tails to yield infective particles capable of DNA transfer.

Asunto(s)

RESUMEN

By using a gel mobility retardation assay, we detected the formation of three major complexes from the binding of nuclear proteins to the promoter of the immunoglobulin lambda 2-chain gene. Two of the complexes were generated by the presence of an unidentified nuclear factor(s) called herein NF-lambda 2. Although the sequences between lambda 2- and lambda 1-chain gene promoters are very similar, the lambda 1-chain promoter did not compete for the binding of NF-lambda 2 efficiently. The binding site of NF-lambda 2 was localized by DNase I footprinting to a 14-bp region which is about 30 bp upstream of the immunoglobulin octamer motif. This region, referred to as the NF-lambda 2 motif, is within an 18-bp region of twofold rotational symmetry. Experiments with oligomers containing either the NF-lambda 2 or the octamer motifs as competitors for binding and DNase I footprinting, showed that the third complex is the product of the simultaneous binding of an octamer-binding protein and NF-lambda 2. Changing the sequence of the NF-lambda 2 motif to that of the lambda 1-chain counterpart abolished the binding ability of NF-lambda 2. Concomitantly, the level of chloramphenicol acetyltransferase expression driven by the mutated lambda 2 promoter decreased by two- to fivefold when compared with that of the wild-type promoter. It is therefore concluded that the interaction of NF-lambda 2 with the NF-lambda 2 motif stimulates transcription of the lambda 2-chain gene.

Asunto(s)

Asunto(s)

RESUMEN

Hybridoma cell lines were transfected with plasmids containing either a rearranged lambda 1 or a rearranged lambda 2 mouse gene. The levels of lambda-chains synthesized by these transfectants were very low or undetectable. Activation of the expression of the lambda 2 gene was achieved artificially by deleting a portion of the region upstream of the promoter. Analogous deletions in the fragment containing the lambda 1 gene did not result in gene activation suggesting that the upstream sequences of lambda 1 and lambda 2 genes have diverged enough to allow differential regulation of their expression. However, both genes were activated by insertion, at a position upstream of the promoter, of a fragment containing the K-chain gene enhancer. These results suggest that the complete set of sequence elements that mediate lambda gene activation during normal B-cell differentiation are not all contained in the fragments of genomic DNA cloned so far, and thus, at least some of them must be located at a considerable distance from the promoter.

Asunto(s)

RESUMEN

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

Asunto(s)

RESUMEN

Bovine papillomavirus-1 (BPV-1) replicates extrachromosomally in certain murine cell lines, suggesting that vectors based on the BPV-1 replicon might provide a means of obtaining more uniform gene expression among independent transformants. We have tested such a vector for the expression in hybridoma cells of the immunoglobulin kappa light-chain gene, but found that the level of expression varies greatly among transformants. Our results also indicate that in these transformants the vector has probably been incorporated into chromosomal DNA.

Asunto(s)

RESUMEN

lambda DNA terminase is the enzyme that catalyses the cleavage of lambda DNA concatemers into genome-size molecules and packages them into the capsid. The cleavage (DNA maturation) takes place in a specific site in the phage DNA called cos. Either one of two Escherichia coli proteins, integration host factor (IHF) and terminase host factor (THF), is required, in addition to terminase, for maturation of wild-type lambda DNA in vitro. In vivo, at least some cos cleavage is known to occur in mutants that are unable to synthesize active IHF. No THF-defective mutants have yet been isolated. In order to determine if IHF, THF or any other host protein is involved in lambda DNA maturation in vivo, I devised a selection for host mutants that are unable to support cos cleavage. The selection is based on the assumption that lambda DNA terminase will kill cells by cleaving chromosomally located cos sites. I found that DNA terminase will indeed kill cells provided that they contain a chromosomal cos site and provided also that they are defective in the host recA or recB genes. These two genes are required for certain pathways of genetic recombination and repair of damaged DNA, and I suggest that they prevent terminase-induced killing by repairing broken chromosomes. Interestingly, mutation in a related host gene, recD, did not render cells susceptible to terminase killing. recD and recB both encode subunits of exonuclease V, but recD mutants, unlike recB, remain proficient in genetic recombination and repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

Lambda DNA packaging in vitro can be examined in stages. In a first step, lambda DNA interacts with terminase to form a DNA-enzyme complex, called complex I. Upon addition of proheads, in a second step, a ternary complex, complex II, containing DNA, terminase and the prohead is formed. Finally, upon addition of the rest of the morphogenetic components, complete phages are assembled. We have investigated the effect of the FI gene product (gpFI) in these reactions and found that a stimulation in phage yield is observed when gpFI is included early in the reaction, at the time when DNA, terminase and proheads interact to form complex II. Measurements of complex II formation revealed that gpFI stimulated the rate of formation of this intermediate. gpFI was further shown to stimulate the addition of proheads to preformed complexes I to give complex II, but the protein did not stimulate complex I formation.

Asunto(s)

RESUMEN

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.

Asunto(s)

RESUMEN

An artificial operon containing the genes coding for the two subunits of lambda DNA terminase, Nul and A, has been constructed. Derivatives of plasmid pBR322 served as the cloning vehicles. The transcription is driven by the pL promoter of phage lambda, and translation of the terminase genes was made efficient by the replacement of the wild-type ribosome-binding sites for those of lambda genes cII and/or D. The operon also carries the oL operator, and this enables regulation of its expression by a thermosensitive repressor. The synthesis of genes Nul and A products is extremely efficient upon derepression. Within 40 min after induction of the operon, the two subunits comprise about 20% of the total cellular protein mass. Crude extracts prepared from these overproducing strains are at least 100 times more active than extracts prepared from induced lambda lysogens in both promotion of lambda DNA packaging and cosmid cleaving. The ability to produce highly concentrated terminase would enormously facilitate the study of its structure and mechanism of action. These extracts are also extremely useful in techniques such as lambda DNA packaging, cosmid mapping and cosmid linearization to improve efficiency of integration into mouse eggs.

Asunto(s)

RESUMEN

Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques.

Asunto(s)