Cleavage by ribonuclease III of the complex of 30 S pre-ribosomal RNA and ribosomal proteins of Escherichia coli

In lysates of Escherichia coli strain AB301-105, newly-formed 30 S pre-ribosomal RNA moved in a complex with protein, at 53 S. A 53 S particle also formed from the purified RNA and 30 S and 50 S ribosomal proteins, and could be cleaved by RNAase III to yield 30 S and 48 S particles.     

Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli ribonuclease III substrates

The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs. The primary agents of dsRNA processing are members of the ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells. Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs. E. coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity. RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes. Some E. coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester. The specific features of the internal loop that establish the pattern of single-strand cleavage are not known. A mutational analysis of the asymmetric [4 nt/5 nt] internal loop of the phage T7 R1.1 substrate reveals that cleavage reactivity is largely independent of internal loop sequence. Instead, the [4/5] asymmetry per se is the primary determinant of cleavage of a single bond within the 5 nt strand of the internal loop. The T7 R1.1 internal loop lacks elements of local tertiary structure, as revealed by sensitivity to cleavage by terbium ion and by the ability of the internal loop to destabilize a small model duplex. The internal loop functions as a discrete structural element in that the pattern of cleavage can be controlled by the specific type of asymmetry. The implications of these findings are discussed in light of RNase III substrate function as a gene regulatory element.     

Characterization of a solvent resistant and thermostable aminopeptidase from the hyperthermophillic bacterium, Aquifex aeolicus

A leucine aminopeptidase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli, and its expression product was purified and characterized. The expressed protein was purified to homogeneity by using heat to denature contaminating proteins followed by ion-exchange chromatography to purify the heat-stable product. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 54 kDa. Kinetic studies on the purified enzyme confirmed that it was a leucine aminopeptidase. The optimum temperature for its activity was around 80 degrees C and the optimum pH was in the range from 8.0 to 8.5. It was stable at high temperatures and 27% of its activity was retained after heating at 115 degrees C for 30 min. The purified enzyme had a pH stability range between 4.0 and 11.0. This aminopeptidase was highly resistant to organic solvents such as methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide, acetone, acetonitrile, dimethyl formamide, 1-propanol, 2-propanol, and dioxane.     

Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III

Members of the ribonuclease III family are the primary agents of double-stranded (ds) RNA processing in prokaryotic and eukaryotic cells. Bacterial RNase III orthologs cleave their substrates in a highly site-specific manner, which is necessary for optimal RNA function or proper decay rates. The processing reactivities of Escherichia coli RNase III substrates are determined in part by the sequence content of two discrete double-helical elements, termed the distal box (db) and proximal box (pb). A minimal substrate of E.coli RNase III, μR1.1 RNA, was characterized and used to define the db and pb sequence requirements for reactivity and their involvement in cleavage site selection. The reactivities of μR1.1 RNA sequence variants were examined in assays of cleavage and binding in vitro. The ability of all examined substitutions in the db to inhibit cleavage by weakening RNase III binding indicates that the db is a positive determinant of RNase III recognition, with the canonical UA/UG sequence conferring optimal recognition. A similar analysis showed that the pb also functions as a positive recognition determinant. It also was shown that the ability of the GC or CG bp substitution at a specific position in the pb to inhibit RNase III binding is due to the purine 2-amino group, which acts as a minor groove recognition antideterminant. In contrast, a GC or CG bp at the pb position adjacent to the scissile bond can suppress cleavage without inhibiting binding, and thus act as a catalytic antideterminant. It is shown that a single pb+db ‘set’ is sufficient to specify a cleavage site, supporting the primary function of the two boxes as positive recognition determinants. The base pair sequence control of reactivity is discussed within the context of new structural information on a post-catalytic complex of a bacterial RNase III bound to the cleaved minimal substrate.     

The hyperthermophilic nature of the metallo-oxidase from Aquifex aeolicus

The stability of the Aquifex aeolicus multicopper oxidase (McoA) was studied by spectroscopy, calorimetry and chromatography to understand its thermophilic nature. The enzyme is hyperthermostable as deconvolution of the differential scanning calorimetry trace shows that thermal unfolding is characterized by temperature values at the mid-point of 105, 110 and 114 degrees C. Chemical denaturation revealed however a very low stability at room temperature (2.8 kcal/mol) because copper bleaching/depletion occur before the unfolding of the tertiary structure and McoA is highly prone to aggregate. Indeed, unfolding kinetics measured with the stopped-flow technique quantified the stabilizing effect of copper on McoA (1.5 kcal/mol) and revealed quite an uncommon observation further confirmed by light scattering and gel filtration chromatography: McoA aggregates in the presence of guanidinium hydrochloride, i.e., under unfolding conditions. The aggregation process results from the accumulation of a quasi-native state of McoA that binds to ANS and is the main determinant of the stability curve of McoA. Kinetic partitioning between aggregation and unfolding leads to a very low heat capacity change and determines a flat dependence of stability on temperature.     

Solution structure of the ribosome recycling factor from Aquifex aeolicus

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.     

Structure and characterization of RNase H3 from Aquifex aeolicus

The crystal structure of ribonuclease?H3 from Aquifex?aeolicus (Aae-RNase?H3) was determined at 2.0?? resolution. Aae-RNase?H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase?H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus?stearothermophilus RNase?H3 (Bst-RNase?H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase?H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase?H3 is relatively long and flexible, while that of Aae-RNase?H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase?H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase?H3 is important for substrate binding, and uses the flat surface of the β-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase?H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.     

The abundant class of nemis repeats provides RNA substrates for ribonuclease III in Neisseriae

About 2% of the Neisseria meningitidis genome is made up by nemis, short DNA sequences which feature long terminal inverted repeats (TIRs). Most nemis are interspersed with single-copy DNA and are found at close distance from cellular genes. In this work, we demonstrate than RNAs spanning nemis of different length and sequence compositions are specifically cleaved at hairpins formed by nemis termini by total cellular lysates derived from both Escherichia coli and Neisseria lactamica strains. The use of cellular extracts from E. coli strains impaired in the activity of known ribonucleases let to establish that cleavage at nemis TIRs is specifically mediated by the endoribonuclease RNase III. Data set the base for the identification of all of the neisserial genes that are regulated by RNase III because of their physical association with nemis DNA.     

Properties of the Glucan Branching Enzyme of the Hyperthermophilic Bacterium Aquifex aeolicus

Abstract

Glucan branching enzymes are responsible for the synthesis of α(1→6) glycosidic bonds in glycogen and amylopectin. The glucan branching enzyme of the hyperthermophile Aquifex aeolicus is the most thermoactive and thermostable glucan branching enzyme described. The gene encoding this glucan branching enzyme was overexpressed in E. coli and purified using γ-cyclodextrin affinity chromatography. Subsequently, the enzyme was stable up to 90°C. Its thermostability may be explained by the relatively high number of aromatic amino acid residues present, in combination with a relatively low number glutamine/asparagine residues. The Km for amylose was 4µM and the Vmax was 4.9 U/mg of protein (at optimal pH and temperature). The side-chain distribution of the branched glucan formed from amylose was determined.     

Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full-length PD are compared with one of these products, an N-terminally truncated protein variant (Delta19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95 degrees C or higher. Delta19PD is more sensitive to temperature effects yielding a half-life of 55 min at 95 degrees C versus 2 h for PD, and values of kcat and Km for prephenate, which are twice those determined for PD at 80 degrees C. Low concentrations of guanidine-HCl activate enzyme activity, but at higher concentrations activity is lost concomitant with a multi-state pathway of denaturation that proceeds through unfolding of the dimer, oligomerization, then unfolding of monomers. Measurements of steady-state fluorescence intensity and its quenching by acrylamide in the presence of Gdn-HCl suggest that, of the two tryptophan residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried tryptophan is at the active site. Tyrosine is a feedback inhibitor of PD activity over a wide temperature range and enhances the cooperativity between subunits in the binding of prephenate. Properties of this thermostable PD are compared and contrasted with those of E. coli chorismate mutase-prephenate dehydrogenase and other mesophilic homologs.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

Split leucine-specific domain of leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus

Leucyl-tRNA synthetase (LeuRS) from Aquifex aeolicus is the only known heterodimer synthetase. It is named LeuRS alphabeta;, and its alpha and beta subunits contain 634 and 289 residues, respectively. Like Thermus thermophilus LeuRS, LeuRS alphabeta has a large extra domain, the leucine-specific domain, inserted into the catalytic domain. The subunit split site is exactly in the middle of the leucine-specific domain and may have a unique function. Here, a series of mutants of LeuRS alphabeta consisting of either mutated alpha subunits and wild-type beta subunits or wild-type alpha subunits and mutated beta subunits were constructed and purified. ATP-PPi exchange and aminoacylation activities and the ability of the mutants to charge minihelix(Leu) were assayed. Interaction of the mutants with the tRNA was assessed by gel shift. Two peptides of eight and nine amino acid residues in the domain located in the alpha subunit were found to be essential for the enzyme's activity. We also showed that the domain in LeuRS alphabeta plays an important role in minihelix(Leu) recognition. Additionally, the domain was found to have little impact on the assembly of the heterodimer, to play a role in the thermal stability of the whole enzyme, and to interact with the cognate tRNA in the predicted manner.     

Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus

RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution. RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.     

Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus

The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.