Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex.

The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr. Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to [32P]p(dT)16 and 70% of the nonspecific binding energy of the intact protein. However, peptide CN6 does not exhibit the binding specificity of the intact protein. Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.     

Genetic mapping of regA mutants of bacteriophage T4D.

SP62, a mutant of bacteriophage T4 shown by Wiberg et al. (1973) to be defective in regulation of T4 protein synthesis, was shown by complementation tests to define a new gene, regA, and by intergenic mapping to lie between genes 43 and 62. The mapping involved crossing SP62 with a quadruple amber mutant defective in genes 42, 43, 62, and 44, selecting all six classes of amber-containing recombinants caused by single crossover events, and then scoring the presence or absence of SP62 in these recombinants. In addition, 15 new, spontaneous regA mutants were isolated, and 13 of these were mapped against each other; a total of eight different mutation sites were thus defined. Most of the new mutants were isolated as pseudorevertants of a leaky amber mutant in gene 62, according to Karam and Bowles (1974), whereas one was identified by virtue of the "white ring" around its plaque, a phenotype possessed by all the regA mutants at high temperature, SP62 was renamed regA1, and the new mutants were named regA2, regA3, etc.     

The bacteriophage T4 regA gene: primary sequence of a translational repressor

The regA gene product of bacteriophage T4 is an autogenously controlled translational regulatory protein that plays a role in differential inhibition (translational repression) of a subpopulation of T4-encoded "early" mRNA species. The structural gene for this polypeptide maps within a cluster of phage DNA replication genes, (genes 45-44-62-regA-43-42), all but one of which (gene 43) are under regA-mediated translational control. We have cloned the T4 regA gene, determined its nucleotide sequence, and identified the amino-terminal residues of a plasmid-encoded, hyperproduced regA protein. The results suggest that the T4 regA gene product is a 122 amino acid polypeptide that is mildly basic and hydrophilic in character; these features are consistent with known properties of regA protein derived from T4-infected cells. Computer-assisted analyses of the nucleotide sequences of the regA gene and its three upstream neighbors (genes 45, 44, and 62) suggest the existence of three translational initiation units in this four-gene cluster; one for gene 45, one for genes 44, 62 and regA, and one that serves only the regA gene. The analyses also suggest that the gene 44-62 translational unit harbors a stable RNA structure that obligates translational coupling of these two genes.     

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.     

Recognition schemes for protein-nucleic acid interactions

The molecular forces involved in protein-nucleic acid interaction are electrostatic, stacking and hydrogen-bonding. These interactions have a certain amount of specificity due to the directional nature of such interactions and the spatial contributions of the steric effects of different substituent groups. Quantum chemical calculations on these interactions have been reported which clearly bring out such features. While the binding energies for electrostatic interactions are an order of magnitude higher, the differences in interaction energies for structures stabilised by hydrogen-bonding and stacking are relatively small. Thus, the molecular interactions alone cannot explain the highly specific nature of binding observed in certain segments of proteins and nucleic acids. It is therefore logical to assume that the sequence dependent three dimensional structures of these molecules help to place the functional groups in the correct geometry for a favourable interaction between the two molecules. We have carried out 2D-FT nuclear magnetic resonance studies on the oligonucleotide d-GGATCCGGATCC. This oligonucleotide sequence has two binding sites for the restriction enzyme Bam H1. Our studies indicate that the conformation of this DNA fragment is predominantly B-type except near the binding sites where the ribose ring prefers a³E conformation. This interesting finding raises the general question about the presence of specificity in the inherent backbone structures of proteins and nucleic acids as opposed to specific intermolecular interactions which may induce conformational changes to facilitate such binding.     

An NMR characterization of the regA protein-binding site of bacteriophage T4 gene 44 mRNA

The conformations of two RNA dodecamers that differ markedly in affinity for the regA protein from bacteriophage T4 have been examined by NMR to see if the ability of that protein to discriminate between mRNAs is based on pre-existing differences in their three-dimensional structures. One of the RNAs examined has the same sequence as the site where regA protein binds when it inhibits the expression of gene 44's mRNA. The second RNA differs from the first in having a U instead of a G at position -9; it binds regA protein 100 times less tightly. The NMR data indicate that both RNAs have similar single-stranded conformations and that they each resemble an isolated strand of a double helix. They also show that most, if not all of the ribose rings in both molecules have appreciable 2'-endo puckering. It is unlikely that regA protein distinguishes between these two molecules on the basis of differences in their global conformations in solution.     

Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions

Bacteriophage T4 gene 32 encodes a single-stranded DNA (ssDNA) binding protein (gp32) required for T4 DNA replication, recombination, and repair. Previous physicochemical studies on gp32 and other ssDNA binding proteins have suggested that binding may involve hydrophobic interactions that result from the close approach of several aromatic amino acid side chains with the nucleic acid bases. In the case of gp32, five tyrosines and two phenylalanines have previously been implicated in gp32.ssDNA complex formation. Site-directed mutagenesis of T4 gene 32 was employed to produce a set of eight gp32 mutant proteins, each of which encoded a single substitution at one of the eight tyrosine residues within gp32. The mutant gp32 proteins were then subjected to physicochemical analysis to evaluate the role of each tyrosine residue in gp32 structure and function. Oligonucleotide binding studies suggest that tyrosine residues 84, 99, 106, 115, and 186 each contribute from 0.3 to 0.7 kcal/mol to ssDNA binding, which corresponds to 3-7% of the overall binding energy for gp32.ssDNA complex formation. Replacement of tyrosine residues 73 and 92 appears to lead to large structural changes that may be the result of disrupting the zinc binding subdomain within gp32.     

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.     

Roles of intrinsic disorder in protein-nucleic acid interactions

Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with them. The interactions of disordered proteins with DNA most often manifest as molding of the protein onto the B-form DNA structure, although some well-known instances involve remodeling of the DNA structure that seems to require that the interacting proteins be disordered to various extents in the free state. By contrast, induced fit in RNA-protein interactions has been recognized for many years-the existence and prevalence of this phenomenon provides the clearest possible evidence that RNA and its interactions with proteins must be considered as highly dynamic, and the dynamic nature of RNA and its multiplicity of folded and unfolded states is an integral part of its nature and function.     

Dynamic protein interactions in the bacteriophage T4 replisome

The bacteriophage T4 DNA replisome is a complex dynamic system employing a variety of proteins to orchestrate the synthesis of DNA on both the leading and lagging strands. Assembly of the protein complexes responsible for DNA synthesis and priming requires the coordination of transient biomolecular interactions. This interplay of proteins has been dissected through the use of small molecules including fluorescent probes and crosslinkers, enabling the development of a complex dynamic structural and kinetic model for DNA polymerase holoenzyme assembly and primosome formation.     

Thermodynamic databases for proteins and protein-nucleic acid interactions

Thermodynamic data regarding proteins and their interactions are important for understanding the mechanisms of protein folding, protein stability, and molecular recognition. Although there are several structural databases available for proteins and their complexes with other molecules, databases for experimental thermodynamic data on protein stability and interactions are rather scarce. Thus, we have developed two electronically accessible thermodynamic databases. ProTherm, Thermodynamic Database for Proteins and Mutants, contains numerical data of several thermodynamic parameters of protein stability, experimental methods and conditions, along with structural, functional, and literature information. ProNIT, Thermodynamic Database for Protein-Nucleic Acid Interactions, contains thermodynamic data for protein-nucleic acid binding, experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data have been incorporated into 3DinSight, an integrated database for structure, function, and properties of biomolecules. A WWW interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. These thermodynamic databases, together with structural databases, help researchers gain insight into the relationship among structure, function, and thermodynamics of proteins and their interactions, and will become useful resources for studying proteins in the postgenomic era.     

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.     

regA protein of bacteriophage T4D: identification, schedule of synthesis, and autogenous regulation.

Proteins labeled with 14C-amino acids after infection of Escherichia coli B by T4 phage were examined by electrophoresis in the presence of sodium dodecyl sulfate. Four regA mutants (regA1, regA8, regA11, and regA15) failed to make a protein having a molecular weight of about 12,000, whereas mutant regA9 did make such a protein; regA15 produced a new, apparently smaller protein that was presumably a nonsense fragment, whereas regA11 produced a new, apparently larger protein. We conclude that the 12,000-dalton protein was the product of the regA gene. The molecular weight assignment rested primarily on our finding that the regA protein had the same mobility as the T4 gene 33 protein, which we identified by electrophoresis of whole-cell extracts of E. coli B infected with a gene 33 mutant, amE1120. Synthesis of wild-type regA protein occurred from about 3 to 11 min after infection at 37 degrees C in the DNA+ state and extended to about 20 min in the DNA- state. However, synthesis of the altered regA proteins of regA9, regA11, and regA15 occurred at a higher rate and for a much longer period in both the DNA+ and DNA- states; thus, the regA gene is autogenously regulated. At 30 degrees C, both regA9 and regA11 exhibited partial regA function by eventually shutting off the synthesis of many T4 early proteins; the specificity of this shutoff differed between these two mutants. We also obtained evidence that the regA protein is not Stevens's "polypeptide 3." As a technical point, we found that, when quantitating acid-precipitable radioactivity in protein samples containing sodium dodecyl sulfate, it was necessary to use 15 to 20% trichloroacetic acid; use of 5% acid, e.g., resulted in loss of over half of the labeled protein.     

Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required.

Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.     

Thermodynamic database for protein-nucleic acid interactions (ProNIT).

MOTIVATION: Protein-nucleic acid interactions are fundamental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein-nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data. Thus, we have developed a Thermodynamic Database for Protein-Nucleic Acid Interactions (ProNIT). RESULTS: We have collected experimentally observed binding data from the literature. ProNIT contains several important thermodynamic data for protein-nucleic acid binding, such as dissociation constant (K(d)), association constant (K(a)), Gibbs free energy change (DeltaG), enthalpy change (DeltaH), heat capacity change (DeltaC(p)), experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data are integrated into a relational database system together with structural and functional information to provide flexible searching facilities by using combinations of various terms and parameters. A www interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. AVAILABILITY: ProNIT is freely accessible at the URL http://www.rtc.riken.go.jp/jouhou/pronit/pronit.html.     

Characterization of the bacteriophage T4 gene 41 DNA helicase

The T4 gene 41 protein and the gene 61 protein function together as a primase-helicase within the seven protein bacteriophage T4 multienzyme complex that replicates duplex DNA in vitro. We have previously shown that the 41 protein is a 5' to 3' helicase that requires a single-stranded region on the 5' side of the duplex to be unwound and is stimulated by the 61 protein (Venkatesan, M., Silver L. L., and Nossal, N. G. (1982) J. biol. Chem. 257, 12426-12434). The 41 protein, in turn, is required for pentamer primer synthesis by the 61 protein. We now show that the 41 protein helicase unwinds a partially duplex DNA molecule containing a performed fork more efficiently than a DNA molecule without a fork. Optimal helicase activity requires greater than 29 nucleotides of single-stranded DNA on the 3' side of the duplex (analogous to the leading strand template). This result suggests the 41 protein helicase interacts with the leading strand template as well as the lagging strand template as it unwinds the duplex region at the replication fork. As the single-stranded DNA on the 3' side of a short duplex (51 base pairs) is lengthened, the stimulation of the 41 protein helicase by the 61 protein is diminished. However, both the 61 protein and a preformed fork are essential for efficient unwinding of longer duplex regions (650 base pairs). These findings suggest that the 61 protein promotes both the initial unwinding of the duplex to form a fork and subsequent unwinding of longer duplexes by the 41 protein. A stable protein-DNA complex, detected by a gel mobility shift of phi X174 single-stranded DNA, requires both the 41 and 61 proteins and a rNTP (preferably rATP or rGTP, the nucleotides with the greatest effect on the helicase activity). In the accompanying paper, we report the altered properties of a proteolytic fragment of the 41 protein helicase and its effect on in vitro DNA synthesis in the T4 multienzyme replication system.