The Nop5-L7A-fibrillarin RNP complex and a novel box C/D containing sRNA of Halobacterium salinarum NRC-1

RNA 2′O-methylation is a frequent modification of rRNA and tRNA and supposed to influence RNA folding and stability. Ribonucleoprotein (RNP) complexes, containing the proteins Nop5, L7A, fibrillarin, and a box C/D sRNA, are guided for 2′O-methylation by interactions of their RNA component with their target RNA. In vitro complex assembly was analyzed for several thermophilic Archaea but in vivo studies are rare, even unavailable for halophilic Archaea. To analyze the putative box C/D RNP complex in the extremely halophilic Halobacterium salinarum NRC-1 we performed pull-down analysis and identified the proteins Nop5, L7A, and fibrillarin and the tRNA^Trp intron, as a typical box C/D sRNA of this RNP complex in vivo. We show for the first time a ribonucleolytic activity of the purified RNP complex proteins, as well as for the RNP complex containing pull-down fractions. Furthermore, we identified a novel RNA (OE4630R-3′sRNA) as part of the complex, containing the typical boxes C/D and C′/D′ sequence motifs and being twice as abundant as the tRNA^Trp intron.     

Interaction of Pseudomonas cytochrome cd1 with the cytoplasmic membrane

Labelling with ferritin-conjugated antibody shows that Pseudomonas cytochrome cd₁ is associated with the inner surface of the cytoplasmic membrane. Cytochrome cd₁ is, however, enriched to the soluble fraction obtained after destruction of Pseudomonas spheroplasts. Comparison of the respiratory nitrite reductase activities, due to this cytochrome, between different cellular fractions and the purified enzyme shows that while the kinetic pattern and the temperature dependence of the activity remain almost the same the molecular activity is enhanced when the enzyme is released from cells.A new assay of respiratory nitrite reductase was developed in this study. The method is based on determination of the stoichiometrical proton consumption accompanying nitrite reduction.     

Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.     

Global Stabilization of rRNA Structure by Ribosomal Proteins S4, S17, and S20

Ribosomal proteins stabilize the folded structure of the ribosomal RNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17, and S20, stabilize the three-dimensional structure of the Escherichia coli 16S 5′ domain. The stability of the complexes was perturbed by varying the concentration of MgCl₂. Each protein influences the stability of the ribosomal RNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5′ domain, while S20 has a more local effect. Multistage folding of individual helices within the 5′ domain shows that each protein stabilizes a different ensemble of structural intermediates that include nonnative interactions at low Mg²⁺ concentration. We propose that the combined interactions of S4, S17, and S20 with different helical junctions bias the free-energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly.     

Small RNAs and Hfq capture unfolded RNA target sites during transcription

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Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Importance of Contact Persistence in Denatured State Loop Formation: Kinetic Insights into Sequence Effects on Nucleation Early in Folding

Protein folding is dependent on the formation and persistence of simple loops early in folding. Ease of loop formation and persistence is believed to be dependent on the steric interactions of the residues involved in loop formation. We have previously investigated this factor in the denatured state of iso-1-cytochrome c using a five-amino-acid insert in front of a unique histidine in the N-terminal region of the protein. Previously, we reported that the apparent pK_a values of loop formation for the most flexible (all Gly) and least flexible (all Ala) insert were, within error, the same. We evaluate whether this observation is due to differences in the persistence of loop contacts or due to effects of local sequence sterics and main-chain hydration on the persistence length of the chain. We also test whether sequence order affects loop formation. Here, we report kinetic results coupled to further mutagenesis of the insert to discern between these possibilities.We find that the amino acid—glycine versus alanine—next to the loop forming histidine has a dominant effect on loop kinetics and equilibria. A glycine in this position speeds loop breakage relative to alanine, resulting in less stable loops. At high percentage of Gly in the insert, rates of loop formation and breakage exactly compensate, leading to a leveling out in loop stability. Loop formation rates also increase with glycine content, inconsistent with poly-Gly segments being more extended than previously suspected due to main-chain hydration or local sterics. Unlike loop breakage rates, loop formation rates are insensitive to local sequence. Together, these observations suggest that contact persistence plays a more important role in defining the “folding code” than rates of loop formation.     

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg²⁺, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s^− 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I_C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.     

Chelation of divalent cations by lomofungin: role in inhibition of nucleic acid synthesis

Lomofungin inhibition of yeast growth and RNA synthesis is prevented by Cu⁺⁺ or Zn⁺⁺ ions which chelate with the antibiotic and prevent its uptake by the cells. EDTA potentiates the inhibition. Mg⁺⁺ ions do not protect $\text{in vivo}$ or against the inhibition of purified bacterial RNA and DNA polymerases. Lomofungin prevents formation of the RNA polymerase. DNA initiation complex, probably by chelation with the firmly bound Zn⁺⁺ of the enzyme.     

Activity and Specificity of the Bacterial PD-(D/E)XK Homing Endonuclease I-Ssp6803I

The restriction endonuclease fold [a three-layer α-β sandwich containing variations of the PD-(D/E)XK nuclease motif] has been greatly diversified during evolution, facilitating its use for many biological functions. Here we characterize DNA binding and cleavage by the PD-(D/E)XK homing endonuclease I-Ssp6803I. Unlike most restriction endonucleases harboring the same core fold, the specificity profile of this enzyme extends over a long (17 bp) target site. The DNA binding and cleavage specificity profiles of this enzyme were independently determined and found to be highly correlated. However, the DNA target sequence contains several positions where binding and cleavage activities are not tightly coupled: individual DNA base-pair substitutions at those positions that significantly decrease cleavage activity have minor effects on binding affinity. These changes in the DNA target sequence appear to correspond to substitutions that uniquely increase the free energy change between the ground state and the transition state, rather than simply decreasing the overall DNA binding affinity. The specificity of the enzyme reflects constraints on its host gene and limitations imposed by the enzyme's quaternary structure and illustrate the highly diverse repertoire of DNA recognition specificities that can be adopted by the related folds surrounding the PD-(D/E)XK nuclease motif.     

Pyrophosphorolysis of CCA addition: implication for fidelity

In nucleic acid polymerization reaction, pyrophosphorolysis is the reversal of nucleotide addition, in which the terminal nucleotide is excised in the presence of inorganic pyrophosphate (PPi). The CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3′ end without using a nucleic acid template. To better understand the reaction mechanism of CCA addition, we tested pyrophosphorolysis of CCA enzymes, which are divided into two structurally distinct classes. Here, we show that only class II CCA enzymes catalyze pyrophosphorolysis and that the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Pyrophosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions. Importantly, pyrophosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3′ end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis. Measurement of kinetic parameters of the class II Escherichia coli CCA enzyme reveals that the enzyme catalyzes pyrophosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to PPi relative to NTP, suggesting a mechanism in which PPi is rapidly released after each nucleotide addition as a driving force to promote the forward synthesis of CCA.     

Crystal Structures of Bacillus subtilis Lon Protease

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.