T4 DNA Replication Complex Research Articles

Mapping Interactions of Single-Stranded (SS) DNA with the SS-DNA Binding Protein (GP32) of the T4 DNA Replication Complex at Specific Nucleotide Residue Positions

The single-stranded (ss)DNA binding protein (gp32) of bacteriophage T4 plays a central role in regulating the functions and integration of the helicase, polymerase and primase components of the T4 DNA replication system. To understand how gp32 interacts with itself and with the other regulatory proteins T4 replication complex, we must first understand the structural details of how this protein binds to ssDNA lattices, both as isolated monomer subunits and as cooperatively bound gp32 clusters. We have explored these issues by monitoring differences in the fluorescence and circular dichroism (CD) spectra of site-specifically positioned monomers and dimer-pairs of 2-aminopurine (2-AP) probes located at various ssDNA positions within the binding site. By mapping spectral changes on binding of gp32 to ssDNA lattices we have been able to characterize interactions at defined positions within the gp32 binding-cleft. We have extended these studies using acrylamide quenching and permanganate foot-printing assays to monitor degrees of base exposure at various lattice positions. Our results show that gp32 binds randomly at low concentrations, and then shifts toward preferential binding at the 5’-ends of the lattice as cooperatively bound gp32 clusters form at higher gp32 concentrations. Bases located near the middle of a gp32 binding site display lower solvent accessibility than those near the ends of the site. These differences in base ‘shielding’ may reflect deeper burial of the middle bases within the electropositive binding cleft, while bases at the ends may be made more accessible by fluctuations of the C- and N-terminal regulatory sub-domains of the protein. Insights into gp32-ssDNA interactions involved in controlling the functions of the T4 DNA replication complex that result from these studies will be discussed.

Using Site Specific Fluorescent Probes to Examine Replication Fork Destabilization by Regulatory Proteins of the Bacteriophage T4 DNA Replication Complex

The reconstituted T4 DNA replication complex serves as the simplest model system to examine the ‘core’ replication mechanisms of higher organisms. In this study we used site-specifically introduced fluorescent base analogues to track local conformational changes of the nucleic acid bases, as well as Cy3/Cy5 dye-pairs inserted into the sugar-phosphate backbones to monitor backbone motions at defined positions within the DNA during interactions of the fork with regulatory proteins. By combining low energy circular dichroism (CD) and fluorescent measurements of the site-specifically introduced fluorescent base analogues with single molecule (sm) FRET experiments with cyanine dyes, we have monitored the binding stoichiometries and interactions of the regulatory proteins with model replication fork constructs. The assembly pathways and binding stoichiometries of gp59 (the helicase loader protein), gp41 (the hexameric helicase) and gp61 (the primase) were studied using functional assays as well as single molecule imaging experiments. The results showed that the stoichiometry of gp59/gp41/gp61 subunits within the functional complex is 1:6:1. CD and fluorescence experiments with base analogue probes show that gp59 preferentially binds to and perturbs the bases at the junction of a forked DNA construct, and that the addition of the gp41 helicase hexamer extends this conformational perturbation deep into the DNA duplex. In contrast, the addition of gp61 perturbs the bases at the fork junction, but not base-pairs deep in the duplex sequence. Based on these results and our previous studies we propose a possible model for DNA duplex unwinding by the helicase loader-primosome-single-strand binding protein complex within the T4 replication system.

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions.

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity.

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding.

Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.

Mechanistic Studies of DNA-Protein Interactions in Bacteriophage T4 DNA Replication Complexes at Single-Base Resolution

Combining biophysical measurements on the function and control of T4 bacteriophage replication complexes with detailed structural information can throw light onto the mechanism of action of these ‘macromolecular machines’. In this study we use the low energy circular dichroism and fluorescent properties of site-specifically introduced fluorescent base analogues to monitor the binding and interactions of gene 32 protein (gp32), the ssDNA binding protein of the T4 DNA replication complex, and gene 59 protein (gp59), the helicase loader protein involved in loading the T4 primosome complex onto replication forks. We show that gp32 binds preferentially to the 3'-end of a short ssDNA oligomer and that only two or three nucleotide bases at the 3' end are directly perturbed by the binding interaction, although the binding site size in the polynucleotide binding mode is 7 nts. Loss of exciton coupling in the CD spectrum and an increase in fluorescence intensity shows that gp32 binding causes extension of the sugar-phosphate backbone of the oligonucleotide. Mechanisms involved in gp32 binding to its ssDNA targets in both the isolated and the cooperative binding modes will be described. In addition our CD and fluorescence experiments with base analogue probes show the gp59 binding to a forked DNA construct perturbs the bases at the junction and suggests that once gp41 is loaded the perturbation extends deep into the double helix. Mechanisms whereby these proteins work in tandem in setting up various functional interactions with the other components of the DNA replication complex will be discussed.

Probing the nucleic acid binding modes of the single‐stranded DNA binding protein of the bacteriophage T4 replication complex

Combining biophysical measurements on the function and control of T4 bacteriophage replication complexes with detailed structural information can throw light onto the mechanism of action of these â€̃macromolecular machines’. In this study we use the low energy circular dichroism and fluorescent properties of site‐specifically introduced fluorescent base analogues to monitor the binding of gene 32 protein (gp32), the ssDNA binding protein of the T4 DNA replication complex, to ssDNA oligomers ranging from 8 to 60 nts in length. We show that gp32 binds preferentially to the 3’‐end of a short ssDNA oligomer and that only two or three nts at the 3’ end are directly involved in the binding interaction. Loss of exciton coupling in the CD spectrum and an increase in fluorescence intensity shows that gp32 binding causes extension of sugar‐phosphate backbone of the oligonucleotide. Further, our results suggest that sliding (or â€̃shuffling’) of the oligonucleotide along the nucleic acid binding cleft of the protein is not observed in the isolated single‐site gp32 DNA binding mode. Mechanisms by which gp32 binds to and covers its ssDNA targets in both an isolated and a cooperative binding mode will be discussed.

Base Pair-Position-Specific DNA ‘Breathing’ At the Replication Fork Junction Regulates Helicase Access

Thermal fluctuations induce transient opening of base pairs in dsDNA constructs. In previous studies with DNA constructs of conserved sequence containing 2-AminoPurine (2-AP) probes, we showed that position-specific base-pair (bp) fraying that depends on proximity to the ss/ds junction can be observed in forked DNA constructs of conserved sequence, and that significant (>1%) thermal fraying of base-pairs at helix ends extends 2-3 bps into the dsDNA. Here we build on these results to study the initial steps of DNA helicases at replication forks. Proteins that bind preferentially to ssDNA can capture thermally frayed bps without the expenditure of chemical (NTP-dependent) free energy. The bacteriophage T4 DNA replication complex provides a favorable model system to study basic helicase mechanisms. The T4 helicase-primase (gp41-gp61) sub-assembly forms a tight-binding helicase that unwinds dsDNA and translocates processively along ssDNA lattices, driven by NTP binding and hydrolysis. We use fluorescence and low energy CD spectral signals of site-specifically placed 2-AP probes to monitor the initial steps of helicase activity at a forked DNA construct. We find, on binding a helicase-primase complex to the DNA construct in the presence of non-hydrolysable NTP, that the first bp on the duplex side of the fork opens and additional destabilization penetrates to ∼ the 3rd bp. This is consistent with a largely passive mechanism for helicase-dependent DNA unwinding, with the helicase complex binding on the 5′→3′ loading strand at the fork and trapping the first adjacent bp as it is opened by thermal fluctuations.

Multiple ATP Binding Is Required to Stabilize the “Activated” (Clamp Open) Clamp Loader of the T4 DNA Replication Complex

Most DNA replication systems include a sliding clamp that encircles the genomic DNA and links the polymerase to the template to control polymerase processivity. A loading complex is required to open the clamp and place it onto the DNA. In phage T4 this complex consists of a trimeric clamp of gp45 subunits and a pentameric loader assembly of four gp44 and one gp62 subunit(s), with clamp loading driven by ATP binding. We measure this binding as a function of input ligand concentration and show that four ATPs bind to the gp44/62 complex with equal affinity. In contrast, the ATPase rate profile of the clamp-clamp loader complex exhibits a marked peak at an input ATP concentration close to the overall Kd (approximately 30 microm), with further increases in bound ATP decreasing the ATPase rate to a much lower level. Thus the progressive binding of the four ATPs triggers a conformational change in the complex that markedly inhibits ATPase activity. This inhibition is related to ring opening by using a clamp that is covalently cross-linked across its subunit interfaces and thus rendered incapable of opening. Binding of this clamp abolishes substrate inhibition of the ATPase but leaves ATP binding unchanged. We show that four ATP ligands must bind to the T4 clamp loader before the loader can be fully "activated" and the clamp opened, and that ATP hydrolysis is required only for release of the loader complex after clamp loading onto the replication fork has been completed.

Mechanistic Studies of the T4 DNA (gp41) Replication Helicase: Functional Interactions of the C-terminal Tails of the Helicase Subunits with the T4 (gp59) Helicase Loader Protein

We compare the activities of the wild-type (gp41WT) and mutant (gp41delta C20) forms of the bacteriophage T4 replication helicase. In the gp41delta C20 mutant the helicase subunits have been genetically truncated to remove the 20 residue C-terminal tail peptide domains present in the wild-type enzyme. Here, we examine the interactions of these helicase forms with the T4 gp59 helicase loader and the gp32 single-stranded DNA binding proteins, both of which are physically and functionally coupled with the helicase in the T4 DNA replication complex. We show that the wild-type and mutant forms of the helicase do not differ in their ability to assemble into dimers and hexamers, nor in their interactions with gp61 (the T4 primase). However they do differ in their gp59-stimulated unwinding activities and in their abilities to translocate along a ssDNA strand that has been coated with gp32. We demonstrate that functional coupling between gp59 and gp41 involves direct interactions between the C-terminal tail peptides of the helicase subunits and the loading protein, and measure the energetics and kinetics of these interactions. This work helps to define a gp41-gp59 assembly pathway that involves an initial interaction between the C-terminal tails of the helicases and the gp59 loader proteins, followed by a conformational change of the helicase subunits that exposes new interaction surfaces, which can then be trapped by the gp59 protein. Our results suggest that the gp41-gp59 complex is then poised to bind ssDNA portions of the replication fork. We suggest that one of the important functions of gp59 may be to aid in the exposure of the ssDNA binding sites of the helicase subunits, which are otherwise masked and regulated by interactions with the helicase carboxy-terminal tail peptides.

Interactions of Bacteriophage T4-coded Primase (gp61) with the T4 Replication Helicase (gp41) and DNA in Primosome Formation

One primase (gp61) and six helicase (gp41) subunits interact to form the bacteriophage T4-coded primosome at the DNA replication fork. In order to map some of the detailed interactions of the primase within the primosome, we have constructed and characterized variants of the gp61 primase that carry kinase tags at either the N or the C terminus of the polypeptide chain. These tagged gp61 constructs have been probed using several analytical methods. Proteolytic digestion and protein kinase protection experiments show that specific interactions with single-stranded DNA and the T4 helicase hexamer significantly protect both the N- and the C-terminal regions of the T4 primase polypeptide chain against modification by these procedures and that this protection becomes more pronounced when the primase is assembled within the complete ternary primosome complex. Additional discrete sites of both protection and apparent hypersensitivity along the gp61 polypeptide chain have also been mapped by proteolytic footprinting reactions for the binary helicase-primase complex and in the three component primosome. These studies provide a detailed map of a number of gp61 contact positions within the primosome and reveal interactions that may be important in the structure and function of this central component of the T4 DNA replication complex.

Interaction of DNA polymerase and DNA helicase within the bacteriophage T4 DNA replication complex. Leading strand synthesis by the T4 DNA polymerase mutant A737V (tsL141) requires the T4 gene 59 helicase assembly protein.

The bacteriophage T4 tsL141 (A737V) mutant in T4 DNA polymerase is temperature-sensitive for DNA replication and an antimutator for some types of mutations. In the accompanying paper (Spacciapoli, P., and Nossal, N. G. (1993) J. Biol. Chem. 269, 438-446), we show that the purified A737V T4 DNA polymerase is less processive than the wild type enzyme as a polymerase, but is more processive as an exonuclease. The bacteriophage T4 multienzyme replication complex reconstituted with the A737V mutant polymerase is defective in both lagging and leading strand synthesis. On lagging strand templates, the A737V polymerase is stimulated by the gene 44/62 and 45 polymerase accessory proteins and the gene 32 DNA binding protein, but is still arrested at pause sites much more frequently than the wild type. In contrast to wild type T4 DNA polymerase, the A737V polymerase does not catalyze leading strand synthesis on a forked duplex template with the polymerase accessory proteins, 32 protein, and the gene 41 protein helicase. The A737V polymerase requires the T4 gene 59 helicase assembly protein, as well as the other proteins, to carry out this reaction. Each of these defects is suppressed by the intragenic L771F mutation that suppresses the antimutator phenotype of the A737V, polymerase in vivo (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140).

Laser cross-linking of proteins to nucleic acids. II. Interactions of the bacteriophage T4 DNA replication polymerase accessory proteins complex with DNA

In this paper we examine the interactions of the polymerase accessory proteins subassembly of the bacteriophage T4 DNA replication complex, using single-pulse ultraviolet laser excitation to induce protein-nucleic acid cross-links. The laser-induced cross-linking permits effective "freezing" of the instantaneous equilibrium state of the complex and thus provides a mechanism to dissect the individual protein-nucleic acid interactions involved in complex assembly. We find that the binding of the gene 44, 62, and 45 proteins is dependent not only on the presence of each of the other proteins, but also on the presence of adenine nucleotide cofactors. We find that the nonhydrolyzable analogs of ATP often behave more like ADP than ATP in these experiments. Gene 45 protein is able to induce an increase in cross-linking of the gp44/62 complex to nucleic acids, and this increased cross-linking correlates with changes in the apparent Km of the gp44/62 complex for polynucleotides and with changes in Vmax during ATP hydrolysis. Our results suggest that the enhanced DNA binding is predominately through the gene 62 protein and not the ATPase catalytic subunit (gene 44 protein). Thus the gene 62 protein seems to play an integral role in gp45-mediated enhancement of the ATP hydrolytic activity of gp44. These results are summarized and integrated in the form of a model for the multiple interactions of the accessory proteins with DNA and one another in the presence of mononucleotide cofactors and substrates.

Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase.

The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of DNA replication. Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32). The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex. We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex. The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32. A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction. These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase. A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43.

“Macromolecular crowding”: thermodynamic consequences for protein-protein interactions within the T4 DNA replication complex.

In vitro biochemical assays are typically performed using very dilute solutions of macromolecular components. On the other hand, total intracellular concentrations of macromolecular solutes are very high, resulting in an in vivo environment that is significantly "volume-occupied." In vitro studies with the DNA replication proteins of bacteriophage T4 have revealed anomalously weak binding of T4 gene 45 protein to the rest of the replication complex. We have used inert macromolecular solutes to mimic typical intracellular solution conditions of high volume occupancy to investigate the effects of "macromolecular crowding" on the binding equilibria involved in the assembly of the T4 polymerase accessory proteins complex. The same approach was also used to study the assembly of this complex with T4 DNA polymerase (gene 43 protein) and T4 single-stranded DNA binding protein (gene 32 protein) to form the five protein "holoenzyme". We find that the apparent association constant (Ka) of gene 45 for gene 44/62 proteins in forming both the accessory protein complex and the holoenzyme increases markedly (from approximately 7 x 10(6) to approximately 3.5 x 10(8) M-1) as a consequence of adding polymers such as polyethylene glycol and dextran. Although the processivity of the polymerase alone is not directly effected by the addition of such polymers to the solution, macromolecular crowding does significantly stabilize the holoenzyme and thus indirectly increases the observed processivity of the holoenzyme complex. The use of macromolecular crowding to increase the stability of multienzyme complexes in general is discussed, as is the relevance of these results to DNA replication in vivo.

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.

Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system.

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.

Crystallization of the gene 45 protein from the DNA replication fork of bacteriophage T4.

The gene 45 protein from bacteriophage T4 has been purified and is crystallized. This protein is part of the T4 DNA replication complex. The crystallized protein is active in complementation assays. X-ray diffraction analysis is in progress; data are measured for the native and several heavy atom derivatives. The crystals diffract to about 3.5-A resolution.

Bacteriophage T4 gene 45. Sequences of the structural gene and its protein product.

Bacteriophage T4 gene 45 codes for a protein whose functions are required for both T4 DNA replication and T4 late gene transcription. To facilitate studies of the interactions of 45 protein with the T4 DNA replication complex and with RNA polymerase, we have determined the primary structure of 45 protein. The amino acid sequence of 45 protein has been determined by correlating nucleotide sequence analysis of gene 45 with protein chemistry studies of 45 protein. Our studies indicate that gene 45 codes for a polypeptide containing 227 amino acids, with a calculated Mr = 24,710. The coding region of gene 46 is preceded by a putative promoter containing sequences which are homologous to Escherichia coli RNA polymerase recognition and binding regions. In addition, there are sequence similarities in the translation initiation regions of gene 45 and the rIIB gene, which may relate to their common regulation by regA protein.