The DnaAcos allele of Escherichia coli: hyperactive initiation is caused by substitution of A184V and Y271H,resulting in defective ATP binding and aberrant DNA replication control

Chromosomal DNA replication is regulated at the level of commitment to this biochemical pathway. In Escherichia coli, DnaA protein appears to regulate this process. A mutant form, DnaAcos, carrying four amino acid substitutions, is apparently defective in responding to regulatory signals, because it induces hyperactive initiation from the bacterial replication origin (oriC). In this report, the phenotype of hyperactive initiation is shown to be the result of two specific amino acid substitutions. One (A184V) immediately adjacent to a Walker A box (P loop motif) causes a defect in ATP binding (Carr and Kaguni, 1996, Mol Microbiol 20: 1307-1318). The second amino acid substitution (Y271H) appears to stabilize the activity of the mutant protein carrying the A184V substitution. The mutant protein carrying both amino acid substitutions (A184V + Y271H) is defective in modulating the frequency of initiation from oriC, as demonstrated by marker frequency analysis of oriC and a locus near the replication terminus. These results indicate that a defect in ATP binding results in aberrant control of DNA replication.     

Nucleotide sequence of the pldB gene and characteristics of deduced amino acid sequence of lysophospholipase L2 in Escherichia coli

The nucleotide sequence of the pldB gene of Escherichia coli K-12, which codes for lysophospholipase L2 located in the inner membrane, was determined. The deduced amino acid sequence of lysophospholipase L2 contains 340 amino acid residues, resulting in a protein with a molecular weight of 38,934. It is characterized by a high content of arginine residues (36 out of 340 residues). The amino acid sequence near the NH2-terminus of the protein is composed of a large number of polar or charged amino acid residues, suggesting that this region cannot be a signal peptide. The hydropathy profile of the deduced amino acid sequence of lysophospholipase L2 was studied. Most of the region was rather hydrophilic, and there was no stretch of hydrophobic amino acid region, such as might be predicted to traverse the lipid bilayer. These results are consistent with the experimental observation that lysophospholipase L2 is extracted by salt solution from the membrane fraction, and it may be classified as a peripheral membrane protein. Computer analysis showed that there is no homology in amino acid sequences between lysophospholipase L2 and other extracellular phospholipases, as well as detergent-resistant phospholipase A, which is another membrane-bound phospholipase in E. coli and whose DNA sequence was determined (Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inoue, K., Ideka, H., Sekiguchi, M., & Nojima, S. (1984) J. Biochem. 96, 1655-1664). This is the first report of the primary structure of a lysophospholipase.     

Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication

We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.     

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.     

The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA

Two species of folate binding protein (FBP), an integral membrane-associated form and a soluble secreted form, have been previously purified from cultured human KB cells. The complete nucleotide sequence of the complementary DNA (cDNA) clone for the coding region of the mature membrane-associated FBP has now been determined, and the deduced amino acid sequence has been computer-analyzed for a prediction of the secondary structure of the protein. The clone has 857 nucleotides of which 678 comprise the coding region for 226 amino acids. The deduced amino sequence contains the identical sequence of the published 18 NH2-terminal amino acids of the purified FBP from KB cells and the published partial amino acid sequence of the human milk FBP except for 1 residue. There was also over 90% homology with the published amino acid sequence of the bovine milk FBP. A total of 16 cysteine residues has been conserved in the three proteins indicating that this amino acid may provide a tertiary structure which is required for its ligand binding function. Northern blot analysis using the cDNA probe identified a single band of 1.28-kilobase pair mRNA in KB cells which was 4.7-fold more intense in folate-depleted cells than in normal cells. These results indicate that the membrane FBP and the soluble FBP in the medium are translation products of the same gene. Computer analysis of the deduced amino acid sequence indicates that there is only one stretch of amino acids of sufficient hydrophobicity and length to span the lipid bilayer of the plasma membrane, but it lacked a predictable helical structure. Those regions of the sequence which did have a predictable helical structure lacked sufficient hydrophobicity required for a membrane anchor. Thus, it is likely that the fatty acids previously reported to be present in the membrane-associated FBP from these cells rather than a peptide sequence provide an important membrane anchoring function.     

Characterization of the oriC region of Mycobacterium smegmatis.

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.     

Mutant DnaA proteins defective in duplex opening of oriC, the origin of chromosomal DNA replication in Escherichia coli

We characterized three mutant DnaA proteins with an amino acid substitution of R334H, R342H and E361G that renders chromosomal replication cold (20 degrees C) sensitive. Each mutant DnaA protein was highly purified from overproducers, and replication activities were assayed in in vitro oriC replication systems. At 30 degrees C, all three mutant proteins exhibited specific activity similar to that seen with the wild-type protein, whereas at 20 degrees C, there was much less activity in a replication system using a crude replicative extract. Regarding the affinity for ATP, the dissociation rate of bound ATP and binding to oriC DNA, the three mutant DnaA proteins showed a capacity indistinguishable from that of the wild-type DnaA protein. Activity for oriC DNA unwinding of the two mutant DnaA proteins, R334H and R342H, was more sensitive to low temperature than that of the wild-type DnaA protein. We propose that R334H and R342H have a defect in their potential to unwind oriC DNA at low temperatures, the result being the cold-sensitive phenotype in oriC DNA replication. The two amino acid residues of DnaA protein, located in a motif homologous to that of NtrC protein, may play a role in the formation of the open complex. The E361 residue may be related to interaction with another protein present in a crude cell extract.     

Cloning and characterization of a single-stranded DNA binding protein that specifically recognizes deoxycytidine stretch.

We previously identified a G-rich silencer element involved in negative regulation of catalase gene expression in some hepatoma cells (Mol. Cell. Biol., (1992), 12, 2525-2533). To study a nuclear binding protein for this element, we screened cDNA libraries from a rat ascites hepatoma cell line by binding with a synthetic oligonucleotide probe and obtained several clones. One of them, designated SW, was studied in detail. A clone (SW2) of this series contained a near full length cDNA encoding a putative peptide with 463 amino acid residues. We isolated this peptide as a fusion protein. It was found that the protein strongly bound to the C-stretch of the DNA sequence in a single strand specific fashion, but absolutely did not to G-rich sequence. The protein bound weakly to the corresponding double-stranded DNA as well as to C-rich RNA sequence. This protein, though not the expected one, was found to be a novel protein whose DNA binding domain was located on the region containing at least 75 amino acid residues of the carboxyl terminus. A proline rich region was also observed in the middle part of the protein. Northern blot profiles indicated extensive and slight expression of both 2.0 kb and 2.7 kb mRNA species in some hepatoma cell lines and in the rat liver, respectively.     

Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.     

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.     

Functional analysis of affinity-purified polyhistidine-tagged DnaA protein.

DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin. We describe a system for efficient production and purification of replicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator. DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with the expected molecular weight. The yield of purified protein was greater than 10 mg per liter of cell culture. The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replication, binding to oriC DNA, binding of allosteric effector adenine nucleotides, and interaction with membrane acidic phospholipids. This system for rapid and high-yield generation of replication-active DnaA protein should facilitate structure-function studies and mutagenic analyses of this initiator protein.     

Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes.

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.     

The carboxy-terminal sequence of the pestivirus glycoprotein E(rns) represents an unusual type of membrane anchor

The E(rns) protein is a structural glycoprotein of pestiviruses that lacks a typical membrane anchor sequence and is known to be secreted from the infected cell. However, major amounts of the protein are retained within the cell and attached to the virion by a so far unknown mechanism. Transient-expression studies with cDNA constructs showed that in a steady-state situation, 16% of the protein is found in the supernatant of the transfected cells while 84% appears as intracellular protein. We show here that E(rns) represents a membrane-bound protein. Membrane binding occurs via the carboxy-terminal region of E(rns). By fusion of this sequence to the carboxy terminus of green fluorescent protein (GFP), the subcellular localization of the reporter protein switched from cytosolic to membrane bound. A core sequence of 11 amino acids necessary for membrane binding was elicited in truncation experiments with GFP constructs. However, this peptide is not sufficient to confer membrane anchoring but needs either upstream or downstream accessory sequences. Analyses with different extraction procedures showed that E(rns) is neither easily stripped from the membrane, like a peripheral membrane protein, nor as tightly membrane bound as a transmembrane protein.     

Role of the amino-terminal region of the DnaA protein in opening of the duplex DNA at the oriC region

We report in this paper that the amino acid residues Ile-26 and Leu-40 of the DnaA protein are essential for the DNA replication activity in vitro. Lines of evidence to support this conclusion are as follows. Variants of the DnaA protein containing either an Ile-26-Ser or Leu-40-Ser replacement were unable to support oriC DNA replication in vitro. Though the mutant DnaA proteins retained the capability to bind oriC DNA, they were unable to open the duplex DNA at oriC. Based on these and other results, we conclude that the N-terminal region of the DnaA protein is involved in the oligomerization of this protein, an essential step for the duplex opening activity at oriC.     

Sequence of gene malG in E. coli K12: homologies between integral membrane components from binding protein-dependent transport systems.

The MalG protein is needed for the transport of maltose in Escherichia coli K12. We present the sequence of gene malG. The deduced amino acid sequence corresponds to a protein of 296 amino acid residues (mol. wt. = 32 188 daltons). This protein is largely hydrophobic (hydrophobic index = 0.83) and is thus presumably an integral inner membrane protein which could span the membrane through six hydrophobic segments. We provide direct evidence from fusion proteins for the translation frame and we also identified the in vitro made MalG protein. We have found a sequence which is highly conserved between MalG and MalF, the other integral inner membrane protein of the maltose transport system. This conserved sequence is also present in all known integral membrane proteins of binding protein-dependent transport systems, always at the same distance (approximately 90 residues) from their COOH terminus. We discuss briefly this finding.     

Replication cycle dependent association of SeqA to the outer membrane fraction of E. coli

The hemimethylated oriC binding activity of the E. coli heavy density membrane fraction (outer membrane) was investigated by DNase I footprinting experiments using membranes obtained from different replication stages of PC-2 (dnaCts) cells. The maximal binding activity was found at the beginning of replication cycle and then decreased gradually. The same pattern of variation was observed with SeqA protein detected in the membranes by immunoblotting. Both binding activity and the presence of SeqA were conserved in the outer membrane even after floating centrifugation of the heavy density membrane fraction in a sucrose gradient, indicating that SeqA in fact can associate with the membrane and that this association varies according to replication cycle. Site specific binding to hemimethylated oriC, of the heavy density membrane obtained from seqA mutant, could be restored by addition of a low amount of His-tagged SeqA protein.     

The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli

After binding to its four 9-mer boxes in the 245-base pair Escherichia coli replication origin (oriC), dnaA protein effects the formation of an "open complex" in an adjacent region made up of three 13-mers (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). This open complex formation requires the ATP form of dnaA protein assisted by HU protein (Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50, 259-265). We now provide direct evidence that dnaA protein binds the 13-mers, sequences that bear no resemblance to the 9-mer box. The evidence is (i) displacement of dnaA protein from the open complex by oriC or by a synthetic oligonucleotide containing the 13-mers, but not by a mutant of oriC lacking the 13-mers; (ii) filter binding of the synthetic (13-mer) oligonucleotide by dnaA protein; and (iii) requirement for the ATP form of dnaA protein assisted by HU protein for temperature-dependent binding to the 13-mer region. Controlled proteolysis of dnaA protein results in a prompt loss of oriC binding; an NH2-terminal 30-kDa peptide contains the domain that binds ATP and phospholipids known to destabilize the tightly bound ATP.     

Cardiolipin activation of dnaA protein, the initiation protein of replication in Escherichia coli

ATP binding to dnaA protein is essential for its action in initiating the replication of plasmids that bear the unique origin of the Escherichia coli chromosome (oriC). ADP bound to that site renders dnaA protein inactive for replication. Diphosphatidylglycerol (cardiolipin), a diacidic membrane phospholipid, displaces the bound nucleotide, and in the presence of components that reconstitute replication, fully reactivates the inert ADP form of dnaA protein. The monacidic phosphatidylglycerol is one-tenth as active as cardiolipin, whereas the neutral phosphatidylethanolamine, the principal E. coli phospholipid, is inactive. Fluphenazine, a tranquilizer drug, blocks cardiolipin activation of dnaA protein, in keeping with the inhibitory action of such agents on phospholipid-dependent enzymes. With the use of this drug to terminate cardiolipin action, dependence of the activation on time, elevated temperature, and high levels of ATP was demonstrated. Cardiolipin binding of nucleotide-free dnaA protein prevents binding of ATP and initiation of oriC replication. Removal of a fatty acid from cardiolipin by phospholipase A reverses this inhibitory effect. The strong and specific interaction of cardiolipin, a cell membrane component, with an essential nucleotide-binding site of dnaA protein, the protein essential for the initiation of chromosome replication, may be an important element in regulating the cell cycle.