The N-Terminal Domain of the Repressor of Staphylococcus aureus Phage Φ11 Possesses an Unusual Dimerization Ability and DNA Binding Affinity

Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors.     

Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators.

The synthesis of the inducible enzyme penicillinase of Bacillus licheniformis is negatively controlled by a repressor (D.A. Dubnau and M.R. Pollock, J. Gen. Microbiol. 41:7-21, 1965; D. J. Sherratt and J. F. Collins, J. Gen. Microbiol. 76:217-230,1973). The molecular organization of the genes coding for penicillinase (penP) and its repressor (penI) has recently been determined (T. Himeno, T. Imanaka, and S. Aiba, J. Bacteriol. 168:1128-1132, 1986). These two genes are transcribed divergently from within a 364-nucleotide region separating the coding sequences. We cloned and sequenced the repressor gene (penIc) from strain 749/C that constitutively produces penicillinase. The penIc and penI+ (wild-type) genes were expressed in Escherichia coli. Complementation analysis indicated that the repressor is the only trans-acting protein required to regulate the expression of the penI and penP genes. We purified the wild-type repressor protein, used it in gel retardation and DNase I protection experiments, and identified three operators positioned in the region between the penP and penI coding sequences. The spatial arrangement of the operators and the hierarchy in repressor binding seen in the protection experiments indicate that (i) the penI gene product represses the expression of the penP gene by physically blocking the RNA polymerase-binding site and (ii) the penI gene is autoregulated.     

Cloning and nucleotide sequence of the penicillinase antirepressor gene penJ of Bacillus licheniformis.

The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid. The penicillinase gene, penP, its repressor gene, penI, and penJ were encoded on the cloned 5.2-kilobase HindIII fragment of the recombinant plasmid pTTE71. The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388). A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site. Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the penI promoter. Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B. licheniformis by chromosomal recombination. The transformant B. licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible. Only when these three genes (penP, penI, and PenJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B. licheniformis. It was concluded that penJ is involved in the penicillinase induction. The regulation of penP expression by penI and penJ is discussed.     

Effects of physical, ionic, and structural factors on the binding of repressor of mycobacteriophage L1 to its cognate operator DNA

To determine the factors influencing the binding of L1 repressor to its cognate operator DNA, several gel shift as well as bioinformatic analyses have been carried out. The data show that time, temperature, salt, and pH each greatly affect the binding. In order to achieve optimum operator binding of L1 repressor in Tris buffer, the minimum requirements of time, temperature, salt, and pH were estimated to be 1 min, 32 degrees C, NaCl (50 mM), and 7.9, respectively. Interestingly Na+ but not NH4+, K+, or Li+ was found to augment significantly the binding activity of CI protein above the basal level. Anions like Cl-, citrate-, acetate-, and H2PO4- do not alter the binding of L1 repressor to its operator. We also show that an in frame deletion mutant of L1 repressor which does not carry the putative HTH motif (at its N-terminal end) fails to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens, etc. Taken together we suggest that N-terminal end of L1 repressor carries a HTH motif. Further analysis of the putative secondary structures of mycobacteriophage repressors reveals that two common regions encompassing more than 90% of primary sequence are present in all the four repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions.     

The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.     

P1 ParB Domain Structure Includes Two Independent Multimerization Domains

ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.     

Temperature-dependent plasmid integration into and excision from the chromosome of Bacillus stearothermophilus

A transformant of Bacillus stearothermophilus carrying a recombinant plasmid, pLP11 (9.5 MDa), on which the penicillinase gene (penP) and kanamycin resistance gene (kan) were located was subjected to mutagenesis, and a mutant plasmid (9.5 MDa; penP kan), designated pTRA117, was obtained. A transformant of B. stearothermophilus carrying pTRA117 could grow at 63 degrees C in medium containing kanamycin, whereas a transformant carrying pLP11 could not. Although pTRA117 was detected as covalently closed circular (ccc) DNA when it was extracted from transformants cultured at 48 degrees C, it was integrated into the host chromosome when the culture temperature was shifted up to 63 degrees C. If the culture temperature was lowered to 48 degrees C from 63 degrees C, a new plasmid (10.7 MDa; penP kan), designated pTRZ117, could be detected as ccc DNA; the size of this plasmid suggested that it was pTRA117 plus a 1.2 MDa DNA fragment of the host chromosome, and this was confirmed by Southern hybridization. pTRZ90 (7.9 MDa; kan) was constructed from pTRZ117 by the deletion of a 2.8 MDa DNA fragment that contained penP. Fresh transformants of B. stearothermophilus that carried either pTRZ117 or pTRZ90 could grow at 65 degrees C.     

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor.

Bacillus licheniformis penicillinase genes, penP and penI, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penI(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48 degrees C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB60E24), the coding region was concluded to be the penI gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penI promoter. The regulation of penP and penI by the repressor is discussed.     

Modification and processing of Bacillus licheniformis prepenicillinase in Escherichia coli. Fate of mutant penicillinase lacking lipoprotein modification site

We have previously shown that Bacillus licheniformis prepenicillinase is modified and processed to form membrane-bound penicillinase in Escherichia coli which contains N-acylglyceride-cysteine27 at the NH2 terminus. In the present study, we have constructed, by in vitro site-directed mutagenesis, two mutant penicillinase genes in which the modification site (the 27th cysteine residue in prepenicillinase) is either converted into serine (penPSer27) or is deleted along with the preceding four residues (Ala23 to Cys27, delta penP2327). The modification, processing, and subcellular localization of these two mutant penicillinases in E. coli cells were studied. Our results indicate that the delta penP2327 deletion mutant prepenicillinase is largely metabolically inert and the unmodified and uncleaved form is associated with the membrane fraction; a small fraction (about 7-9%) appears to contain glyceride-modified prepenicillinase (presumably at the Cys-21 position) which is not cleaved. In contrast, the Cys-27 in equilibrium Ser-27 point mutant prepenicillinase is processed into two forms which contain Asn-29 and Ser-35 at their NH2 termini, respectively, and the bulk of the processed penicillinase appears to be located in the peri-plasm. These results are discussed in terms of the substrate specificities of signal peptidases in E. coli.     

Identification of a novel region of the cytoplasmic Dynein intermediate chain important for dimerization in the absence of the light chains

Cytoplasmic dynein is the multisubunit protein complex responsible for many microtubule-based intracellular movements. Its cargo binding domain consists of dimers of five subunits: the intermediate chains, the light intermediate chains, and the Tctex1, Roadblock, and LC8 light chains. The intermediate chains have a key role in the dynein complex. They bind the three light chains and the heavy chains, which contain the motor domains, but little is known about how the two intermediate chains interact. There are six intermediate chain isoforms, and it has been hypothesized that different isoforms may regulate specific dynein functions. However, there are little data on the potential combinations of the intermediate chain isoforms in the dynein complexes. We used co-immunoprecipitation analyses to demonstrate that all combinations of homo- and heterodimers of the six intermediate chains are possible. Therefore the formation of dynein complexes with different combinations of isoforms is not limited by interaction between the various intermediate chains. We further sought to identify the domain necessary for the dimerization of the intermediate chains. Analysis of a series of truncation and deletion mutants showed that a 61-amino-acid region is necessary for dimerization of the intermediate chain. This region does not include the N-terminal coiled-coil, the C-terminal WD repeat domain, or the three different binding sites for the Tctex1, LC8, and Roadblock light chains. Analytical gel filtration and covalent cross-linking of purified recombinant polypeptides further demonstrated that the intermediate chains can dimerize in vitro in the absence of the light chains.     

Structural characterization and corepressor binding of the Escherichia coli purine repressor.

The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein.     

The C-terminal region of the exosome-associated protein Rrp47 is specifically required for box C/D small nucleolar RNA 3'-maturation

Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6. Expression of the N-terminal domain of Rrp47 in yeast complemented most RNA-processing defects associated with the rrp47Δ mutant but failed to complement the defect observed in 3'-end maturation of box C/D small nucleolar RNAs. Consistent with these results, protein capture assays revealed an interaction between the C-terminal region of Rrp47 and the small nucleolar ribonucleoproteins Nop56 and Nop58. Filter binding assays demonstrated that deletion of the lysine-rich sequence at the C terminus of Rrp47 blocked RNA binding in vitro. Furthermore, a protein mutated both at the C terminus and within the N-terminal domain showed a synergistic defect in RNA binding without impacting on its ability to interact with Rrp6. These studies provide evidence for a role of Rrp47 in registering a small nucleolar ribonucleoprotein particle assembly, functionally characterize the Sas10/C1D domain of Rrp47, and show that both the C terminus of Rrp47 and the N-terminal domain contribute to its RNA-binding activity.     

Regulatory functions of the N-terminal domain of the 70-kDa subunit of replication protein A (RPA)

Replication protein A (RPA) is the major single-stranded DNA-binding protein in eukaryotes. RPA is composed of three subunits of 70, 32, and 14 kDa. The N-terminal domain of the 70-kDa subunit (RPA70) has weak DNA binding activity, interacts with proteins, and is involved in cellular DNA damage response. To define the mechanism by which this domain regulates RPA function, we analyzed the function of RPA forms containing a deletion of the N terminus of RPA70 and mutations in the phosphorylation domain of RPA (N-terminal 40 amino acids of the 32-kDa subunit). Although each individual mutation has only modest effects on RPA activity, a form combining both phosphorylation mimetic mutations and a deletion of the N-terminal domain of RPA70 was found to have dramatically altered activity. This combined mutant was defective in binding to short single-stranded DNA oligonucleotides and had altered interactions with proteins that bind to the DNA-binding core of RPA70. These results indicate that in the absence of the N-terminal domain of RPA70, a negatively charged phosphorylation domain disrupts the activity of the core DNA-binding domain of RPA. We conclude that the N-terminal domain of RPA70 functions by interacting with the phosphorylation domain of the 32-kDa subunit and blocking undesirable interactions with the core DNA-binding domain of RPA. These studies indicate that RPA conformation is important for regulating RPA-DNA and RPA-protein interactions.     

Molecular determinants of the interaction between coxsackievirus protein 3A and guanine nucleotide exchange factor GBF1

The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum-to-Golgi complex transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (Arf1), thereby interfering with Arf1-mediated COP-I recruitment. This study was undertaken to gain more insight into the molecular determinants underlying the interaction between 3A and GBF1. Here we show that 3A mutants that have lost the ability to dimerize are no longer able to bind to GBF1 and trap it on membranes. Moreover, we identify a conserved region in the N terminus of 3A that is crucial for GBF1 binding but not for 3A dimerization. Analysis of the binding domain in GBF1 showed that the extreme N terminus, the dimerization/cyclophilin binding domain, and the homology upstream of Sec7 domain are required for the interaction with 3A. In contrast to that of full-length GBF1, overexpression of a GBF1 mutant lacking its extreme N terminus failed to rescue the effects of 3A. Together, these data provide insight into the molecular requirements of the interaction between 3A and GBF1.     

A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.     

Primer utilization by DNA polymerase alpha-primase is influenced by its interaction with Mcm10p

Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains. In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S. pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not. These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates. A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex. We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication.