Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25°C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.     

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.     

Prediction of three-dimensional structure of Escherichia coli ribosomal RNA

A model for the tertiary structure of 23S, 16S and 5S ribosomal RNA molecules interacting with three tRNA molecules is presented using the secondary structure models common to E. coli, Z. mays chloroplast, and mammalian mitochondria. This ribosomal RNA model is represented by phosphorus atoms which are separated by 5.9 A in the standard A-form double helix conformation. The accumulated proximity data summarized in Table 1 were used to deduce the most reasonable assembly of helices separated from each other by at least 6.2 A. Straight-line approximation for single strands was adopted to describe the maximum allowed distance between helices. The model of a ribosome binding three tRNA molecules by Nierhaus (1984), the stereochemical model of codon-anticodon interaction by Sundaralingam et al. (1975) and the ribosomal transpeptidation model, forming an alpha-helical nascent polypeptide, by Lim & Spirin (1986), were incorporated in this model. The distribution of chemically modified nucleotides, cross-linked sites, invariant and missing regions in mammalian mitochondrial rRNAs are indicated on the model.     

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.     

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.     

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.     

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.