Divergence of the mRNA targets for the Ssb proteins of bacteriophages T4 and RB69
© Borjac-Natour et al; licensee BioMed Central Ltd. 2004
Received: 12 July 2004
Accepted: 17 September 2004
Published: 17 September 2004
The single-strand binding (Ssb) protein of phage T4 (T4 gp32, product of gene 32) is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination. It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets. Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding. We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains. In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins. In translation assays, we show that RB69 gp32 is, like T4 gp32, an autogenous translational repressor. We also show that the natural mRNA targets (translational operators) for the 2 proteins are diverged in sequence from each other and yet can be repressed by either gp32. Results of chemical and RNase sensitivity assays indicate that the gp32 mRNA targets from the 2 related phages have similar structures, but differ in their patterns of contact with the 2 repressors. These and other observations suggest that a range of gp32-RNA binding specificities may evolve in nature due to plasticity of the protein-nucleic acid interaction and its response to modulation by the C-terminal domain of this translational repressor.
KeywordsSsb protein, gp32, RNA-binding proteins, DNA-binding proteins translational control, DNA replication
T4 gp32, the single-strand binding (Ssb) protein of bacteriophage T4, is a well studied member of the Ssb protein family, and was the first such ssDNA-binding replication protein to be discovered . The protein, product of T4 gene 32, is an essential component of the phage DNA replication complex and also plays essential roles in DNA repair and recombination [2, 3]. Like other Ssb proteins, T4 gp32 facilitates transactions at the replication fork, especially along the lagging strand, through its binding to the unwound DNA template and its specific interactions with other protein components of the DNA replisome. T4 gp32 is known to stimulate the phage induced DNA polymerase (T4 gp43) and to play a role in the dynamics of primosome (T4 gp61-gp41 complex) recruitment by the primase-helicase assembly protein T4 gp59 [4–6]. In general, Ssb proteins lack specificity to the ssDNA sequence and this property allows them to perform their physiological roles at all genomic locations undergoing replication, repair or recombination. The presence of a Ssb protein in the right place at the right time may depend, in large measure, on specificity of its interactions with other proteins from the same biological source.
T4 gp32 has the interesting property of being able to control its own biosynthesis at the translational level in vivo. The protein binds to a specific target (translational operator) in the 5' leader segment of the mRNA from gene 32, and represses translation of this RNA . Another Ssb protein, gp5 of the M13 ssDNA phage family, has also been shown to act as a mRNA-specific translational repressor, although in this case, the RNA target is located in the message for another essential M13 replication protein, gp2 (an endonuclease) [8, 9]. It is not known if other Ssb proteins, especially those for cellular DNA replication and maintenance, also possess RNA binding functions that regulate specific translation or other physiologically important RNA-dependent processes. In T4, the physiological link between the sequence-independent ssDNA and specific RNA binding functions of gp32 has been explained by a model based on in vitro measurements of the protein's binding affinities to different nucleic acid ligands. It has been observed that ssDNA is favored over translational operator RNA as a ligand for T4 gp32 and that RNA of nonspecific sequence is the least preferred nucleic-acid ligand for this Ssb protein [10–12]. In vivo, T4 encoded mRNA for gp32 is intrinsically more metabolically stable than the typical prokaryotic mRNA and is thought to have opportunities to undergo many cycles of gp32-mediated repression and depression during the replication and other processing of phage DNA. The potential for translation of this mRNA in the T4 infected E coli host is thought to be determined by availability of ssDNA in the metabolic pool [10, 13, 14]. DNA damage or unwinding transactions are thought to draw gp32 away from its mRNA target to the exposed ssDNA, thus causing derepression of translation and upward adjustments in gp32. Repression of the mRNA would then be reestablished if the amount of gp32 exceeded the number of exposed ssDNA sites for the protein. This model is consistent with many in vivo observations relating to levels of T4 gp32 biosynthesis under conditions of DNA damage or abnormal accumulation of ssDNA in the phage infected bacterial host .
It is not clear how T4 gp32 distinguishes between specific RNA and the non-specific nucleic acid sequence of ssDNA or ssRNA ligands. It appears that single-strandedness of the nucleic acid is not the most important criterion used by the protein to selectively bind its own message in the phage-induced mRNA pool. The translational operator for T4 gp32 has been mapped by RNA footprinting assays and determined to consist of two contiguous components, a 5' terminal ~28-nucleotide component that forms a folded structure (RNA pseudoknot) and an adjacent, less structured, >40-nucleotide component that lies 3' to the pseudoknot [15, 16]. The 3' terminal component includes several repeats of UUAAA or UAAA sequences, in addition to harboring typical prokaryotic nucleotide determinants for translation initiation by ribosomes [7, 16, 17]. The RNA pseudoknot and UUAAA/UAAA elements are both essential for autogenous repression of the mRNA by T4 gp32 [15, 16, 18]. In vitro studies suggest that the pseudoknot serves as the initial recognition (nucleation) site for the protein and that this gp32-RNA interaction leads to cooperative binding of additional gp32 monomers to the less structured downstream sequence containing the UUAAA/UAAA elements and ribosome-binding site (RBS) . Cooperative binding to the mRNA is envisaged to be analogous to gp32-ssDNA interactions, except that the UUAAA/UAAA sequence elements probably contribute to specificity of the mRNA interaction to the protein.
The 3-dimensional structure of intact T4 gp32 has not been solved, although a number of biochemical and physiological observations have provided clues that the protein is modularly organized into 3 distinct domains . In particular, studies with proteolytic fragments of purified T4 gp32, including the analysis of a crystal structure for one of these fragments , have assigned the ssDNA binding function to a module formed by an internal segment of the 301-residue protein. It is presumed that this domain is responsible for binding specific RNA as well, although no direct evidence exists for this notion. In the studies described here, we show that the ssDNA-binding domain is highly conserved between T4 gp32 and the phylogenetic variant of this protein from the T4-like phage RB69. Yet, we also show that sequences of the mRNA targets for the two Ssb proteins are different and that the two repressors differ in their patterns of interaction with these targets. We present results suggesting that specificity of gp32 to RNA has co-evolved with specificity of this Ssb protein to other phage induced proteins of DNA metabolism that interact with gp32's C-terminal domain. Our studies suggest that the ability of a diverging regulatory RNA to make alternate contacts with a mutually plastic, but highly conserved, RNA-binding protein site may allow the RNA to tolerate mutational changes without loss of the regulatory function. Such plasticity of the interacting partners could allow for the evolution of a broad spectrum of gp32-RNA binding specificities despite selective pressures that conserve the amino acid sequence of the protein's nucleic acid-binding domain.
Bacterial and phage strains used
The E coli K-12 strain K802 (hsdR, hsdM+, gal, met, supE) was used as host in cloning experiments and the E coli B strain NapIV (hsdR k +, hsdM k +, hsdS k +, thi, sup o ) was the host for plasmid-mediated gene expression studies that utilized lambda pL control. E coli B strain BL21(DE3), which harbors a T7 RNA polymerase gene under cellular lac promoter control , was used as the host for T7 Φ10-promoter plasmids in pilot experiments that assessed toxicity of cloned RB69 gene 32 to bacterial cells.
Cloning and nucleotide sequence determination of RB69 gene 32
Assays for plasmid directed gene 32 expression
Purification of gp32 from clones of the structural gene
RB69 gp32 and T4 gp32 were purified from the overproducing clones pRBg32Δop (RB69 gp32) and pYS69 (T4 gp32), respectively. We used the gp32 purification protocol outlined by Bittner et al,  with minor modifications. The preparation of crude extracts, from 6-liter batches of heat-inducible E coli NapIV clones of phage genes, was as described previously for T4 RegA protein . Anionic-exchange chromatography (using Q-Sepharose; Cat# 17-0510-01; Pharmacia) was as described for purification of plasmid-generated RB69 gp43 . Under the conditions used, gp32 eluted at 0.3–0.4 M NaCl. In the subsequent chromatographic step, utilizing Phenyl-Sepharose (Cat#17-0965-05; Pharmacia), we tested column fractions for nuclease contamination by incubating 4 μl samples with plasmid DNA (~1 μg) overnight at room temperature and then analyzing the mixtures by agarose gel electrophoresis. The gp32-containing fractions that exhibited no hydrolysis of the plasmid DNA were pooled and the protein was purified further by chromatography on ssDNA-agarose (Cat #15906-019; Invitrogen). Pooled fractions from the ssDNA chromatography were dialyzed against a gp32 storage buffer containing 0.1 M NaCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM DTT and 50% glycerol Protein stocks (at 4–8 mg gp32/ml) were stored at -20°C until used.
Preparation of RNA for in vitro studies
RNA preparations used for footprinting and other in vitro studies originated from in vitro transcription of pSP72 clones of the desired gene 32 sequences Methods have been described elsewhere . Phage-specific RNA sequences of the purified transcription products used for footprinting included nucleotide positions -102 to +161 (relative to the initiator AUG) in case of the RB69 gene 32 transcripts and positions -96 to +161 in case of the T4 gene 32 transcripts. These products also included a 10-nt sequence from the plasmid's T7 promoter region RNA sequencing was carried out by using the RVT-catalyzed primer-extension (cDNA synthesis) method described elsewhere [23, 29]. Sequencing primers were annealed to codons 12 to 20 of the transcripts and the sequenced segments of the RNA spanned nucleotide positions +36 through about -100 relative to the initiator AUG. For in vitro translation assays, the RNA preparations included full length and truncated versions of the gene 32 open-reading frame from each of the 2 phage sources.
Assays for gp32-mediated in vitro translational repression
We used E coli S30 cell-free extracts (Cat#L1020; Promega) with purified pSP72-based gene 32 recombinant DNA (coupled transcription-translation assays) or purified RNA (DNA-free translation assays) to assess repressor activities of purified RB69 gp32 and T4 gp32. With plasmid-directed gene 32 expression, it was possible to use expression of the plasmid borne bla gene (β-lactamase) as an internal control. Each 50 μl in vitro assay reaction mixture (placed in a 15-ml conical tube) contained 1 μg of plasmid DNA template or 4 μg RNA, 5 μl of a mixture of all amino acids (1 mM each) except L-methionine, 1 μl of an S30-premix cocktail (containing rNTPs, tRNAs, an ATP generating system and required salts), 15 μl S30 extract and the balance of volume in nuclease-free water Reaction mixtures, including any added gp32, were constituted in an ice bath before transferring to 37°C for incubations (30 or 60 min). Reactions were stopped by rechilling in the ice bath. Proteins from 5 μl samples were precipitated with 20 μl acetone, collected by centrifugation, dried and suspended in SDS extraction buffer for analysis by SDS-PAGE and autoradiography. Analysis of plasmid encoded (N-terminal) gp32 fragments was carried out in SDS-PAGE (10% gels) using Tricine as the electrophoresis buffer. This buffer system allows for effective resolution of small polypeptides . When used, purified gp32 was added at concentrations ranging between 5 and 20 μM.
Treatments of RNA with RNases and chemical agents
The RNA-modifying chemical reagents Dimethylsulfate (DMS; Cat# D18,630-9; Aldrich) and Diethylpyrocarbonate (DEPC; Cat# D5758; Sigma) and the ribonucleases (RNases A1, T1 and V1 respectively) were used to probe RB69- and T4-derived operator RNAs for intrinsically structured regions. The RNases were also used for RNA footprinting (protection by gp32) studies.
DMS was diluted in absolute ethanol at ratios of 1:2, 1:4, and 1:5 ratio v/v and its effects were analyzed at the three concentrations. The reaction buffer contained 30 mM HEPES pH 7.5, 10 mM MgCl2. Reactions were stopped in 0.5 M β-mercaptoethanol and 0.75 M sodium acetate. The protocol for DEPC treatment was identical to that for DMS, except that we used 1 μl of DEPC per 100 μl of reaction mix and incubated the reactions at room temperature for 10 min.
For the RNase-sensitivity assays, including gp32-mediated RNA footprinting, digestions with RNases A1 and T1 were carried out in 30 μl buffer containing 60 mM NH4Cl, 10 mM Mg acetate, 10 mM Tris-HCl pH 7.4, and 6 mM β-Mercaptoethanol. The buffer for digestions with RNase V1 contained 25 mM Tris-HCl pH 7.2, 10 mM MgCl2, and 0.2 M NaCl Incubations were at 37°C in 30 μl buffer in all cases. RNase treatments were halted with an equal volume of buffer containing 0.4 M Na acetate pH 5.2, 20 mM EDTA, and 30 μg E coli tRNA. When used for RNA footprinting, RB69 gp32 or T4 gp32 was added at concentrations in the range between 1 μM and 5 μM.
A sequence comparison between T4 gp32 and RB69 gp32
The RB69 IC59-32 region
RB69 gp32 and T4 gp32 are functionally similar
Figure 4 shows results from experiments that measured the effects of RB69 gp32 on its own synthesis in vivo (Fig 4A) and in vitro (Fig 4B). The in vivo experiments measured plasmid-directed RB69 gene 32 expression by E coli clones carrying wild-type and mutant versions of the RB69 gene. As shown in Fig 4A, induced expression of the gene was lower (by ~4-fold) with the wild-type construct than with deletion mutants of the untranslated 5' leader of the mRNA (RBG32Δop, Fig 4A) or missense mutants in the structural gene from this phage (PL2 and PL8 constructs; Fig 4A). These observations are consistent with the explanation that RB69 gp32, like T4 gp32, is able to bind and repress its own mRNA. The results shown in Fig 4B confirm that purified RB69 gp32 is a potent repressor of translation of purified mRNA for this protein.
We have used similar experiments to those for Fig 4 to compare repressor activities of T4 gp32 and RB69 gp32 on identical RNA targets, and observed that either protein can repress gene 32-specific mRNA from either source (results not shown). However, such experiments, which require 10–30 μM purified protein to demonstrate repression (Fig 4B), did not unambiguously distinguish between the RNA-binding specificities of the 2 proteins. Also, in phage-plasmid complementation assays, we observed that the cloned RB69 wild-type gene 32 supported efficient growth of T4 gene 32 mutants (bursts of ~100) By these criteria, the T4 and RB69 proteins appeared to be similarly functional in each other's physiological systems. Yet, the natural targets for the 2 proteins are clearly different from each other in topography (Fig 3) and as we describe later, RNA-binding specificity differences between the 2 proteins could be detected through in vitro RNA-footprinting assays, which utilized lower concentrations of gp32 than is usually required to detect gp32-mediated repression by in vitro translational assays.
RNA structure in the RB69 gene 32 translational initiation region (TIR)
The footprints of T4 gp32 and RB69 gp32 on gene 32-specific RNA targets from T4 and RB69
1. At the protein concentrations used (1–5 μM), the RB69 gp32 footprint on the RNA target from RB69 was 5 residues longer than the footprint of this protein on the T4-derived RNA target; however, the positions of the 2 footprints relative to the respective initiator AUG and 5' terminal boundary of the pseudoknot structure appeared to be identical (Fig 6)
2. As can be seen in Figs 7A and 7B, RB69 gp32 protected its own mRNA target strongly within the nucleotide segment between U(-14) and G(-61), and weakly in the segment from U(-2) to G(-9) In contrast, as seen in Figs 7C and 7D, T4 gp32 protected this RNA strongly only in the segment from C(-42) to G(-61)
3. As can be seen in Fig 8C and 8D, T4 gp32 protected the T4-derived RNA strongly in the G(+3) to U(-70) segment In contrast, RB69 gp32 protected this RNA target best in the U(-16) to U(-70) segment (Fig 7A and 7B)
It should be noted that the gp32 footprint sizes reported here are shorter than has been reported in studies that utilized higher concentrations of T4 gp32 with T4-specific RNA targets [16, 32]. As stated earlier in this report (Fig 4), the higher gp32 concentrations (>5 μM) mask specificity differences between the T4 and RB69 proteins.
Phages T4 and RB69 are phylogenetically related to each other and encode homologous sets of DNA replication proteins that exhibit a significant degree of compatibility with each other's biological systems [22, 24]. Despite such overlaps in function, we have commonly observed specificity differences between protein homologues from the 2 phage systems. For example, in plasmid-phage complementation assays, RB69 DNA polymerase (gp43) was observed to be just as effective as T4 gp43 in T4 DNA replication in vivo, whereas the T4 enzyme was less effective than its RB69 counterpart for RB69 DNA replication [22, 33]. Also, the 2 DNA polymerases, like the 2 Ssb proteins compared here, are RNA-binding autogenous translational repressors that differ in RNA binding specificity and RNA target sequence. Studies with the T4 versions of gp43 and gp32 clearly show that the binding of these proteins to specific RNA is mutually exclusive with their binding to DNA [7, 34]. So, conservation of the translational functions of these proteins may be related to conservation of their replication functions. Based on previous studies with RB69 gp43 , as well as the current study with RB69 gp32, we surmise that neither of these translational repressors possesses a domain that binds RNA exclusively. Rather, in both cases, the RNA binding site seems to be contained within the region of the protein that binds DNA. Thus, it is possible that in phage infected cells, specific RNA serves as a regulator of both the biosynthesis and replicative activities of these proteins.
In the purified system we have used to compare RNA footprints for gp32 from T4 and RB69 (Figs 6, 7, 8), we observed that the same RNA target could exhibit different patterns of protection depending on source of the Ssb protein. This observation suggests that the RNA-protein interaction is intrinsically flexible and can accommodate a range of RNA sequences as long as these sequences can be made to assume a certain configuration. In addition, the interaction could be subject to modulation by intra- and intermolecular protein-protein interactions of the repressor. In this regard, it is known that the extreme N-terminal segment (~20 residues) and C-terminal segment (~100 residues) of T4 gp32 have profound effects on the ssDNA binding activity, which is housed in the region bracketed by these 2 segments of the protein [19, 36, 37]. The N-terminal segment determines cooperative binding to ssDNA (through gp32-gp32 interactions) and the C-terminal segment has been implicated in interactions of gp32 with other phage induced proteins . Possibly, the observed sequence divergence between the C-terminal domains of T4 gp32 and RB69 gp32 (Fig 2) was in part coupled to divergence of the mRNA targets during evolution of the 2 related translational repressors.
Although we cannot rule out the possibility that the C-terminal domain of gp32 influences specificity to RNA by interacting directly with this ligand, there are indications that this negatively charged segment of gp32 is a modulator of gp32 interactions with nucleic acids rather than a carrier of nucleic acid binding determinants. In particular, a small deletion that maps within this protein segment (Δ32PR201; Fig 2) exhibits altered specificity to other proteins but has no effects on autogenous control of gp32 synthesis in vivo . Also, recent studies with purified RB69 gp32 implicated the C-terminal domain of this protein in regulating access of gp43 from the same phage to binding sites in the ssDNA-binding module of the Ssb protein . It has also been shown that in T4, the ssDNA-binding module of gp32 forms specific crosslinks to gp59, the phage-induced primase-helicase loading protein [41, 42]. Such observations suggest that the 2 nucleic-acid binding functions of gp32 may be subject to regulation by a combination of intra- and intermolecular protein-protein interactions involving the divergence-prone C-terminal domain. It would be particularly interesting to find out if the gp32 sequence divergence near the DNA binding residue Y106 (Fig 2) is important for RNA recognition. X-ray crystallographic studies  suggest that T4 gp32 residues T101-K110 constitute part of the ssDNA-binding surface of the protein, which includes Y84, Y99, Y106 and the nearby "LAST" motif (residues 110–114; 31). Also, as suggested by the 3D structure, these residues are located within or very close to the Zn-binding domain of the protein; ie, the putative "zinc-finger" sequence Cys77-X3-His-X5-Cys-X2-Cys90, which has counterparts in a number of RNA-binding proteins . The construction and analysis of RB69-T4 gp32 chimeras could help to establish if the divergence near Y106 is responsible for the observed differences in RNA footprints between T4gp32 and RB69 gp32 (Figs 6, 7, 8).
In summary, we envisage that as a mediator of gp32's interactions with other phage induced proteins, the C-terminal domain of gp32 may co-diverge with its protein targets to maintain mutual recognition, and that structural plasticity of a conserved ssDNA-binding domain may allow an also diverging RNA target to establish rearranged contacts within a relatively conserved protein pocket. It is unclear if the 2 sets of divergence are interconnected, but together, they could facilitate the evolution of a high degree of diversity in how the synthesis and/or replication activity of this Ssb protein is regulated among phylogenetic relatives of T4. It will be important to find out if this diversity includes RNA ligands for gp32 that control the DNA-binding activity but not synthesis of gp32, or if autogenous translational repression has been replaced by other mechanisms for control of gene 32 in some T4 relatives. There is at least one reported example where evolution resulted in lack of RNA binding function in the Ssb protein of an M13-like phage . Also, a scan of available genomic sequences for T4-like phages http://phage.bioc.tulane.edu/ reveals a high degree of sequence divergence in the putative translational operator regions of the corresponding gene 32 regions. In one case, phage RB49 (GenBank NC_005066), it has been reported that there are no indications that an RNA pseudoknot structure exists in the putative TIR for gene 32, although the UUAA/UUAAA sequence units are conserved in the RB49 IC59-32 region . It remains to be seen if gp32 from this and other T4 like phages that appear to lack the RNA pseudoknot do bind their respective TIR regions or repress their own translation.
Finally, we should comment about ORF32.1 (Fig 1) and its possible relevance to evolution of the mRNA target for gp32. This ORF is present in some T4-like genomes (eg T4 and GenBank Ac No AF033323) and absent in others (eg, RB69 and GenBank Ac No AY310907). Recently, it was shown that T4 ORF 32.1 encodes a Seg-type (G1Y-YIG family) homing endonuclease (now named SegG) that mediates its own transfer, along with T4 gene 32, to the ORF32.1-less genome of phage T2 in T4 × T2 genetic crosses. We note that the 5' terminal sequence of the RNA pseudoknot for T4 gp32 translational control overlaps the reading frame of the segG gene, in addition to being very similar (~83% identity) to the corresponding segment of the pseudoknot sequence of RB69, which lacks a segG gene (Fig 3). Possibly, this portion of the RNA pseudoknot preexisted the entry of an ORF32.1-like sequence element into the gene 59-32 intercistronic region of a T4 progenitor and that the modern day segG gene (ORF32.1) may be a chimera consisting of an extension of the parental segG reading frame into the recipient genome's pseudoknot sequence. Such lateral transfer events and subsequent mutation may have profound influences on evolution of the RNA binding functions of proteins that have relaxed sequence but stringent structural requirements for their RNA target.
All the experimental work reported here was carried out by the first author in partial completion of the requirements for the PhD in Biochemistry. This author's work was supported by NIH grant GM54627 and a predoctoral stipend from Tulane University School of Medicine. The second and corresponding authors were also supported by NSF grant 038236 during preparation of this manuscript. We thank our colleagues James Nolan and Henry Krisch for stimulating discussions about phage evolutionary biology and Jill Barbay for extensive help with manuscript preparation.
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