RNA chaperone activity and RNA-binding properties of the E. coli protein StpA

The E. coli protein StpA has RNA annealing and strand displacement activities and it promotes folding of RNAs by loosening their structures. To understand the mode of action of StpA, we analysed the relationship of its RNA chaperone activity to its RNA-binding properties. For acceleration of annealing of two short RNAs, StpA binds both molecules simultaneously, showing that annealing is promoted by crowding. StpA binds weakly to RNA with a preference for unstructured molecules. Binding of StpA to RNA is strongly dependent on the ionic strength, suggesting that the interactions are mainly electrostatic. A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity. This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA. We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.     

Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro.

An Escherichia coli gene, stpA, has been identified and cloned based on its ability to suppress the Td- phenotype of a resident, splicing-defective phage T4 td (thymidylate synthase) gene. The stpA gene, which was localized to 60.24 min on the E. coli chromosome, encodes a 15.3-kDa protein. Overproduction of StpA in vivo led to an increase in td pre-mRNA levels and modest enhancement of td mRNA:pre-mRNA ratios. Consistent with its in vivo effect, purified StpA promoted RNA splicing in vitro, and facilitated RNA annealing and strand exchange with model substrates. These results suggest that StpA promotes splicing of the intron by binding RNA nonspecifically, resolving misfolded precursor molecules and facilitating association of critical base pair elements. Furthermore, proteinase K treatment of StpA-assembled precursors prior to the initiation of the splicing reaction still resulted in splicing enhancement, indicating that StpA is not required for the catalytic step, unlike the Neurospora splicing effector CYT-18, whose presence was necessary for catalysis to proceed. Together these results suggest that StpA has chaperone activity in vitro, with the property of promoting assembly of the precursors into an active conformation, in contrast to splicing effectors that stabilize the catalytically active intron structure.     

The effect of an Escherichia coli regulatory mutation on transfer RNA structure.

The trpX mutation in Escherichia coli reduces trp operon attenuation in strains carrying wild-type tRNA^Trp. The trpX^? phenotype is alleviated (attenuation is restored) in UGA-suppressor tRNA^Trp-carrying strains (Yanofsky &; Soll, 1977).The tRNA from various trpX^? strains was characterized biochemically. Sequence analyses of wild-type tRNA^Trp and UGA suppressor tRNA^Trp, both derived from trpX^? strains, reveal an unmodified A in the position (adjacent to the anticodon) normally occupied by the hypermodified base ms²i⁶A.In addition, several tRNAs from trpX^? cells were characterized by RPC-5 column chromatography. We find that only tRNAs normally having ms²i⁶A exhibit altered elution profiles when compared to the homologous tRNAs from trpX^? cells. Introduction of the UGA suppressor into trpX^? cells does not restore normal Chromatographic behavior. These results suggest that the trpX gene product is necessary for the synthesis of ms²i⁶A. Thus, we propose that miaA (for the first gene involved in ms²i⁶A synthesis) replaces the trpX designation.The results reported here are discussed with regard to a model proposed by Lee &; Yanofsky (1977) in which efficient translation of the tandem trp codons in the leader sequence RNA is required for normal attenuation of the trp operon.     

Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.     

Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism

The ygfZ gene product of Escherichia coli represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the E. coli protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.     

Importance of the conserved nucleotides around the tRNA-like structure of Escherichia coli transfer-messenger RNA for protein tagging

A bacterial RNA functioning as both tRNA and mRNA, transfer-messenger RNA (tmRNA) rescues stalled ribosomes and clears the cell of incomplete polypeptides. For function, Escherichia coli tmRNA requires an elaborate interplay between a tRNA-like structure and an internal mRNA domain that are connected by a 295 nt long compact secondary structure. The tRNA-like structure is surrounded by 16 unpaired nt, including 10 residues that are >95% conserved among the known 140 tmRNA sequences. All these residues were mutated to define their putative role(s) in trans-translation. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all variants. As anticipated from the low sequence conservation, mutating positions 8–12 and position 15 affects neither aminoacylation nor protein tagging. Mutating a set of two conserved positions 13 and 14 abolishes both functions. Probing the solution conformation indicates that this defective mutant adopts an alternate conformation of its acceptor stem that is no more aminoacylatable, and thus inactive in protein tagging. Selected point mutations at the conserved nucleotide stretches 16–20 and 333–335 seriously impair protein tagging with only minor changes in their solution conformations and aminoacylation. Point mutations at conserved positions 19 and 334 abolish trans-translation and 70S ribosome binding, although retaining nearly normal aminoacylation capacities. Two proteins that are known to interact with tmRNA were purified, and their interactions with the defective RNA variants were examined in vitro. Based on phylogenetic and functional data, an additional structural motif consisting of a quartet composed of non-Watson–Crick base pairs 5′-YGAC-3′:5′-GGAC-3′ involving some of the conserved nucleotides next to the tRNA-like portion is proposed. Overall, the highly conserved nucleotides around the tRNA-like portion are maintained for both structural and functional requirements during evolution.     

Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives.

Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.     

Mannose permease of Escherichia coli. Domain structure and function of the phosphorylating subunit

The mannose permease of Escherichia coli is a component of the phosphotransferase system. It transports mannose and related hexoses by a mechanism that couples sugar transport with sugar phosphorylation. It is a complex consisting of two transmembrane subunits (II-PMan and II-MMan) and a hydrophilic subunit (IIIMan). IIIMan also exists in a soluble form as dimer in the cytoplasm. Each monomer of IIIMan consists of two structurally and functionally distinct domains which are linked by a flexible hinge of the sequence KAAPAPAAAAPKAAPTPAKP. Both domains are transiently phosphorylated. The NH2-terminal domain (P13) is phosphorylated at N-3 of His-10 by the cytoplasmic phosphorylcarrier protein phospho-HPr. The COOH-terminal domain (P20) is phosphorylated by P13 at N-1 of His-175. Phosphoryltransfer occurs not only between P13 and P20 on the same IIIMan subunit but also between isolated domains and between domains on different subunits of the dimer. In the presence of the IIMan subunits, the phosphoryl group is directly transferred from His-175 of P20 to the sugar substrates of the permease. The P13 domain contains the contact sites for dimerization of IIIMan. The P20 domain contains the contact sites for interaction with the IIMan subunits. By reconstructing the ptsL gene, the two domains were expressed as individual polypeptides and the length of the hinge between P13 and P20 was changed. The in vivo and in vitro activities of mutant IIIMan were little affected by these modifications. The hinge is highly sensitive to proteolytic cleavage in vitro and its specificity for proteases can be modified by introducing the appropriate specificity determinants.     

Crystal structure of the Escherichia coli RNA degradosome component enolase.

The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A. There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules, flexible loops close onto the active site. This closure mimics the conformation of the substrate-bound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a specific role to it, a structural role is possible.     

Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.     

Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein

CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a nucleic acid processing system in bacteria and archaea that interacts with mobile genetic elements. CRISPR DNA and RNA sequences are processed by Cas proteins: in Escherichia coli K-12, one CRISPR locus links to eight cas genes (cas1, 2, 3 and casABCDE), whose protein products promote protection against phage. In the present paper, we report that purified E. coli Cas3 catalyses ATP-independent annealing of RNA with DNA forming R-loops, hybrids of RNA base-paired into duplex DNA. ATP abolishes Cas3 R-loop formation and instead powers Cas3 helicase unwinding of the invading RNA strand of a model R-loop substrate. R-loop formation by Cas3 requires magnesium as a co-factor and is inactivated by mutagenesis of a conserved amino acid motif. Cells expressing the mutant Cas3 protein are more sensitive to plaque formation by the phage λvir. A complex of CasABCDE ('Cascade') also promotes R-loop formation and we discuss possible overlapping roles of Cas3 and Cascade in E. coli, and the apparently antagonistic roles of Cas3 catalysing RNA-DNA annealing and ATP-dependent helicase unwinding.     

Proteolytic activity of YibP protein in Escherichia coli

Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A yibP disrupted mutant grows in rich medium at 37 degrees C but not at 42 degrees C. In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42 degrees C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate beta-casein.