Polyethylene glycol molecular crowders enhance the catalytic ability of bimolecular bacterial RNase P ribozymes

Abstract

The modular structure of bacterial ribonuclease P (RNase P) ribozymes, which recognize tertiary structures of precursor tRNAs (pre-tRNAs) to cleave their 5′ leader sequence, can be dissected physically into the two structured domain RNAs (S-domain and C-domain). Separately prepared S-domain RNA and C-domain RNA assemble to form bimolecular forms of RNase P ribozymes. We analyzed the effects of polyethylene glycols (PEGs) on pre-tRNA cleavage catalyzed by bimolecular RNase P ribozymes to examine the effects of molecular crowding on the reaction. PEG molecular crowders significantly enhanced the activities of bimolecular RNase P ribozymes, some of which were hardly active without PEGs.     

Osservazioni Preliminari sui Sistemi Degradanti dell'RNA nei semi di Ricino

Abstract

Preliminary observations on the enzymatic degradation of RNA in castor bean seeds. — Cocucci, Maggio, Monroy and Marrè have shown the decrease of RNA content during ripening in castor bean seeds, and its increase during germination. Furthermore, these Authors have demonstrated that in the dry ripe seeds the ribosomes are undetectable, and that they increase rapidly during germination. Two peaks of ribosomes are easily detected upon ultracentrifugal analysis in germinating seeds (Cocucci and Sturani). These observations were the basis for our investigations of the enzymes of RNA metabolism in castor bean seeds. This paper deals with our preliminary observations on RNA degrading enzymes in these tissues. We have been able to measure RNase activity, phosphodiesterase, 3′-,5′- and 2′-nucleotidases in castor bean seeds at different stages of development. RNase activity (measured in crude extracts) changes little during the ripening process, its rate corresponding to 40–50 μMoles of nucleotides liberated from RNA per hour and per gram of fresh weight. In the dry seeds, RNase activity is 30–40 μMoles of nucleotides/h.g.f.w., and it increases to about 60–70 μMoles/h/g.f.w. after 72 hours of germination.

Phosphodiesterase activity is about 4–5 μMoles/h.g.f.w.

The following rates have been found in seeds almost completely ripe seeds for 3′-, 5′- and 2′-nucleotidase activities, respectively 45–50 μMoles/h.g.f.w.; 6–7 μMoles/h.g.f.w.; 8 μMoles/h.g.f.w.; ATP-ase activity was of about 80–100 μMoles of phosphate liberated /h.g.f.w. - The high activity of 3′-nucleotidase, of the same order of that of RNase, suggests that these two enzymes are responsible for degradation of RNA to nucleosides and inorganic phosphate. Further investigations are being carried on to define the biochemical properties of castor bean RN-ase.     

Crystallographic Study of Mechanism of Ribonuclease Ti-Catalysed Specific RNA Hydrolysis

Abstract

Ribonuclease T₁ (RNase T₁) cleaves the phosphodiester bond of RNA specifically at the 2′-end of guanosine. 2′-guanosinemonophosphate (2′-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T₁. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T₁ is globular in shape and consists of a 4.5 turns α-helix lying “below” a four-stranded antiparallel β-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2′-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C=O, guanine O₆ and N₁H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T₁.guanine recognition.     

Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

Small stable RNA maturation and turnover in Bacillus subtilis

Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis‐specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5′ and 3′ extremities, respectively, whereas the RNase P RNA is matured on its 3′ side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co‐translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3′ processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.     

Tracking the elusive 5′ exonuclease activity of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> RNase J

Key message

Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5′ exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation.

Abstract

RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5′ end maturation is thought to be achieved by the combined action of a 5′ exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5′ exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5′ exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5′ exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.

     

Mechanism of DNA chain growth. XI. Structure of RNA-linked DNA fragments of Escherichia coli

The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ-³²P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the ³²P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …     

Synthesis and Properties of Oligonucleotide Chimeras Containing 5′-Amino-2′-deoxy-2′-fluoroarabinonucleosides

Abstract

Oligonucleotide analogues comprised of 2′-deoxy-2′-fluoro-β-D-arabinose units joined via P3′-N5′ phosphoramidate linkages (2′F-ANA^5′N) were prepared for the first time. Among the compounds prepared were a series of 2′OMe-RNA-[GAP]-2′OMe-RNA ‘chimeras’, whereby the “GAP” consisted of DNA, DNA^5′N, 2′F-ANA or 2′F-ANA^5′N segments. The chimeras with the 2′F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5′-amino derivatives, i.e., 2′F-ANA > DNA > 2′F-ANA^5′N > DNA^5′N. Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E.coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2′F-ANA > 2′F-ANA^5′N > DNA^5′N. The corresponding relative rates observed with the human enzyme were: gap DNA > 2′F-ANA^5′N > 2′F-ANA > DNA^5′N.     

Modes of Binding of 2′-AMP to RNase T1 A Computer Modeling Study

Abstract

The modes of binding of adenosine 2′-monophosphate (2′-AMP) to the enzyme ribonuclease (RNase) T₁ were determined by computer modelling studies. The phosphate moiety of 2′-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites - (1) The primary base binding site where the guanine of 2′-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3′-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2′-AMP to RNase T₁ were found to be of nearly the same energy implying that in solution 2′-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T₁ - 2′-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T₁ catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2′-AMP and 2′-GMP with RNase T₁ reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme - 2′-GMP complex.     

Boranophosphate Nucleic Acids - A Versatile DNA Backbone

Abstract

Important chemical and biochemical properties of boranophosphate DNA and RNA oligonucleotides are reviewed. Stereoregular boranophosphate oligomers can be synthesized enzymatically and form stable duplexes with DNA. Fully boronated, non-stereoregular oligothymidylates, synthesized chemically, form hybrids with poly(A) that have lower melting points than oligothymidylate:poly(A), yet they nevertheless can support the RNase H mediated cleavage of RNA.     

Reassignment of the active site histidines in ribonuclease A by selective deuteration studies.

The C₂H resonance of the active site histidine residue designated AS-2, which has the lower pK_a of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP-3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS-2 is absent in the spectrum of RNase S′ reconstituted using S-peptide deuterated at the C₂ of His 12, and in that of the RNase S′-CMP-3′ complex. We thus demonstrate that histidine AS-2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C₂H resonances made earlier by other authors.     

E. COLI RNASE HI AND THE PHOSPHONATE-DNA/RNA HYBRID: MOLECULAR DYNAMICS SIMULATIONS

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3′–O–CH₂–P–O–5′ or 3′–O–P–CH₂–O–5′) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3′-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg⁺⁺ · 4H₂O chelate complex (bound in the active site) were analyzed in detail. Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).     

Determinants in the rpsT mRNAs recognized by the 5′‐sensor domain of RNase E

RNase E plays a central role in processing virtually all classes of cellular RNA in many bacterial species. A characteristic feature of RNase E and its paralogue RNase G, as well as several other unrelated ribonucleases, is their preference for 5′‐monophosphorylated substrates. The basis for this property has been explored in vitro. At limiting substrate, cleavage of the rpsT mRNA by RNase E (residues 1–529) is inefficient, requiring excess enzyme. The rpsT mRNA is cleaved sequentially in a 5′ to 3′ direction, with the initial cleavage(s) at positions 116/117 or 190/191 being largely driven by direct entry, independent of the 5′‐terminus or the 5′‐sensor domain of RNase E. Generation of the 147 nt 3′‐limit product requires sequential cleavages that generate 5′‐monophosphorylated termini on intermediates, and the 5′‐sensor domain of RNase E. These requirements can be bypassed with limiting enzyme by deleting a stem‐loop structure adjacent to the site of the major, most distal cleavage. Alternatively, this specific cleavage can be activated substantially by a 5′‐phosphorylated oligonucleotide annealed 5′ to the cleavage site. This finding suggests that monophosphorylated small RNAs may destabilize their mRNA targets by recruiting the 5‐sensor domain of RNase E ‘in trans’.     

A Solid-Phase Synthesis of 2′,5′-Linked Oligoadenylates (2-5A)

Abstract

2′,5′-Oligoadenylate 5′-triphosphates (2-5A) as products of 2-5A synthetase and activators of ribonuclease L (RNase L), are mediators in one of the mechanisms of interferon′s antiviral action. Upon activation, RNase L inhibits protein synthesis due to the degradation of RNAs. This activity of 2-5A could possibly find an application in virus or cancer chemotherapy, but two major barriers prevent the use of 2′,5′-linked oligoadenylates as therapeutic agents. The 2-5A is readily degraded by a 2′,5′ phosphodiesterase and as a highly negatively charged molecule, is not readily taken up by cells. One possible solution to this latter limitation might be found in chemical modifications of the 2-5A structure. Many analogues of 2-5A have been already obtained with modified base, ribose or phosphate moieties. While these have provided some important information about the enzyme- activator interactions, the cell permeability problem still remains unsolved. One of the major obstacles in this study is lack of a convenient method of synthesis of 2′,5′ ribonucleotides of widely varying structure.     

Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1

Abstract

The mechanism of action of ribonuclease (RNase) T₁ is still a matter of considerable debate as the results of x-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes of RNase T₁ and some of its mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T₁ with the reaction intermediate guanosine 2′, 3′-cyclic phosphate (G>p). The puckering of the guanosine ribose moiety in the minimum energy conformer of the RNase T₁ - GpC (substrate) complex was found to be O4′-endo and not C3′-endo as in the RNase T₁ - 3′-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action of RNase T₁ has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T₁ - GpC and RNase T₁ - G>p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.     

The mode of action of divalent cations on the RNase catalyzed hydrolysis of RNA and uridylyl (3'-5') uridine

Pseudo first order rate constants (k′) have been measured for the RNase A catalyzed hydrolysis of uridylyl (3′–5′) uridine at several ionic strengths and compositions. The k′ values are independent of Mg²⁺ concentration between 0 and 10 mM. This shows that for hydrolysis of RNA, in which Mg²⁺ concentration does change k′, the perturbation must be through binding of Mg²⁺ to the substrate RNA rather than to the enzyme RNase.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.