Ribonucleotide Residues Research Articles

Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity

Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5’-end interactions. Unconventional interactions of PliAgo with the 5’-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.

Ribonucleotides and RNA Promote Peptide Chain Growth

AbstractAll known forms of life use RNA‐mediated polypeptide synthesis to produce the proteins encoded in their genes. Because the principal parts of the translational machinery consist of RNA, it is likely that peptide synthesis was achieved early in the prebiotic evolution of an RNA‐dominated molecular world. How RNA attracted amino acids and then induced peptide formation in the absence of enzymes has been unclear. Herein, we show that covalent capture of an amino acid as a phosphoramidate favors peptide formation. Peptide coupling is a robust process that occurs with different condensation agents. Kinetics show that covalent capture can accelerate chain growth over oligomerization of the free amino acid by at least one order of magnitude, so that there is no need for enzymatic catalysis for peptide synthesis to begin. Peptide chain growth was also observed on phosphate‐terminated RNA strands. Peptide coupling promoted by ribonucleotides or ribonucleotide residues may have been an important transitional form of peptide synthesis that set in when amino acids were first captured by RNA.

Ribonucleotides and RNA Promote Peptide Chain Growth.

All known forms of life use RNA-mediated polypeptide synthesis to produce the proteins encoded in their genes. Because the principal parts of the translational machinery consist of RNA, it is likely that peptide synthesis was achieved early in the prebiotic evolution of an RNA-dominated molecular world. How RNA attracted amino acids and then induced peptide formation in the absence of enzymes has been unclear. Herein, we show that covalent capture of an amino acid as a phosphoramidate favors peptide formation. Peptide coupling is a robust process that occurs with different condensation agents. Kinetics show that covalent capture can accelerate chain growth over oligomerization of the free amino acid by at least one order of magnitude, so that there is no need for enzymatic catalysis for peptide synthesis to begin. Peptide chain growth was also observed on phosphate-terminated RNA strands. Peptide coupling promoted by ribonucleotides or ribonucleotide residues may have been an important transitional form of peptide synthesis that set in when amino acids were first captured by RNA.

Structural Impact of Single Ribonucleotide Residues in DNA.

Single ribonucleotide intrusions represent the most common nonstandard nucleotide type found incorporated in genomic DNA, yet little is known of their structural impact. This lesion incurs genomic instability in addition to affecting the physical properties of the DNA. To probe for structural and dynamic effects of single ribonucleotides in various sequence contexts-AxC, CxG, and GxC, where x=rG or dG-we report the structures of three single-ribonucleotide-containing DNA duplexes and the corresponding DNA controls. The lesion subtly and locally perturbs the structure asymmetrically on the 3' side of the lesion in both the riboguanosine-containing and the complementary strand of the duplex. The perturbations are mainly restricted to the sugar and phosphodiester backbone. The ribose and 3'-downstream deoxyribose units are predominately in N-type conformation; backbone torsion angles ϵ and/or ζ of the ribonucleotide or upstream deoxyribonucleotide are affected. Depending on the flanking sequences, the C2'-OH group forms hydrogen bonds with the backbone, 3'-neighboring base, and/or sugar. Interestingly, even in similar purine-rG-pyrimidine environments (A-rG-C and G-rG-C), a riboguanosine unit affects DNA in a distinct manner and manifests different hydrogen bonds, which makes generalizations difficult.

ClaRNA: a classifier of contacts in RNA 3D structures based on a comparative analysis of various classification schemes.

The understanding of folding and function of RNA molecules depends on the identification and classification of interactions between ribonucleotide residues. We developed a new method named ClaRNA for computational classification of contacts in RNA 3D structures. Unique features of the program are the ability to identify imperfect contacts and to process coarse-grained models. Each doublet of spatially close ribonucleotide residues in a query structure is compared to clusters of reference doublets obtained by analysis of a large number of experimentally determined RNA structures, and assigned a score that describes its similarity to one or more known types of contacts, including pairing, stacking, base–phosphate and base–ribose interactions. The accuracy of ClaRNA is 0.997 for canonical base pairs, 0.983 for non-canonical pairs and 0.961 for stacking interactions. The generalized squared correlation coefficient (GC2) for ClaRNA is 0.969 for canonical base pairs, 0.638 for non-canonical pairs and 0.824 for stacking interactions. The classifier can be easily extended to include new types of spatial relationships between pairs or larger assemblies of nucleotide residues. ClaRNA is freely available via a web server that includes an extensive set of tools for processing and visualizing structural information about RNA molecules.

RNA Bricks—a database of RNA 3D motifs and their interactions

The RNA Bricks database (http://iimcb.genesilico.pl/rnabricks), stores information about recurrent RNA 3D motifs and their interactions, found in experimentally determined RNA structures and in RNA–protein complexes. In contrast to other similar tools (RNA 3D Motif Atlas, RNA Frabase, Rloom) RNA motifs, i.e. ‘RNA bricks’ are presented in the molecular environment, in which they were determined, including RNA, protein, metal ions, water molecules and ligands. All nucleotide residues in RNA bricks are annotated with structural quality scores that describe real-space correlation coefficients with the electron density data (if available), backbone geometry and possible steric conflicts, which can be used to identify poorly modeled residues. The database is also equipped with an algorithm for 3D motif search and comparison. The algorithm compares spatial positions of backbone atoms of the user-provided query structure and of stored RNA motifs, without relying on sequence or secondary structure information. This enables the identification of local structural similarities among evolutionarily related and unrelated RNA molecules. Besides, the search utility enables searching ‘RNA bricks’ according to sequence similarity, and makes it possible to identify motifs with modified ribonucleotide residues at specific positions.

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-β) expression

The human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene is unusual as it contains an editing site where nontemplated ribonucleotide residues can be inserted. This RNA editing can lead to the expression of the viral P, PD, putative W, and theoretical V protein from a single gene. Although the HPIV3 PD protein has been detected, its function and those of the W and V proteins are poorly understood. Therefore, we first used reverse genetics techniques to construct and rescue a recombinant (r)HPIV3 clone with a polyhistidine sequence at the 5' end of the P gene for tagged protein detection. Western blot analysis demonstrated the presence of the P, PD, and W proteins, but no V protein was detected. Then, we functionally studied the D domain of the PD protein by constructing two rHPIV3 knockout clones that are deficient in the expression of the D domain. Results from growth kinetic studies with infected MA-104 and A596 cells showed that viral replication of the two knockout viruses (rHPIV3-ΔES and rHPIV3-ΔD) was comparable to that of the parental virus in both cell lines. However, viral mRNA transcription and genomic replication was significantly reduced. Furthermore, cytokine/chemokine profiles of A549 cells infected with either knockout virus were unchanged or showed lower levels compared to those from cells infected with the parental virus. These data suggest that the D domain of the PD protein may play a luxury role in HPIV3 RNA synthesis and may also be involved in disrupting the expression of beta interferon.

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

The (3'-->5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 degrees C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 degrees C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 degrees C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 degrees C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.

A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome: predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA

Human respiratory syncytial virus (HRSV) escape mutants selected with antibodies specific for the attachment (G) protein contain diverse genetic alterations, including point mutations, premature stop codons, frame shift changes and A to G hypermutations. The latter changes have only been found in mutants selected with antibodies directed against the conserved central region of the G protein. This gene segment fulfils substrate requirements for adenosine deaminases that act on RNA (ADARs): i.e. it is an A+U rich region of 137 residues, and 98 or 106 of them--for A/Mon/3/88 or Long HRSV strains, respectively--are predicted to form intramolecular base pairs leading to a stable RNA secondary structure. In addition, when sequences of the G gene from natural isolates are compared in terms of pairwise substitutions, A to G+G to A changes are preferentially observed in regions where stable intramolecular dsRNA secondary structures are predicted to occur. In this study, a model is proposed in which, in addition to nucleotide misincorporations, reiterative A to G changes in HRSV are generated by ADAR activity operating in short segments (100-200 ribonucleotide residues) of the HRSV genome with high tendency for intramolecular base pairing.

3'-5' Exonucleolytic activity of DNA polymerases: structural features that allow kinetic discrimination between ribo- and deoxyribonucleotide residues.

We have determined rates for the excision of nucleotides from the 3' termini of chimeric DNA-RNA oligonucleotides using the Klenow fragment (KF) and two other DNA polymerases, from phages T4 and T7. For these studies, we synthesized DNA-RNA chimeric oligonucleotides with RNA residues in defined positions. When a ribonucleotide residue was placed at the 3' terminus, all three DNA polymerases removed it at the same rate as they did for substrates composed solely of deoxynucleotide residues. There was a decrease in the excision rate, however, when a ribonucleotide residue was located at the second or third position from the 3' terminus. When both the second and third positions were occupied by ribonucleotide residues, the excision rate for the 3' terminal nucleotide was reduced even further and was almost identical to the rate observed when the DNA polymerases encountered single-stranded RNA. The magnitude of the effect of ribonucleotide residues on the excision rate was lower when Mn(2+) replaced Mg(2+) as the essential divalent cation. Two KF mutations, Y423A and N420A, selectively affected the excision rates for the chimeric substrates. Specifically, Y423A totally abolished the rate reduction when there was a single ribonucleotide residue immediately preceding the 3' terminus, whereas N420A diminished, but did not eliminate, the rate reduction relative to that of wild-type KF when the single ribonucleotide residue occupied either the second or third position from the 3' terminus. These results are consistent with the structure of a KF-ss DNA complex from which it can be deduced, by modeling, that a 2' OH group on the second sugar from the 3' terminus would sterically clash with the Tyr 423 side chain, and a 2' OH group on the third sugar would clash with the side chain of Asn 420. The corresponding mutations in T4 DNA polymerase did not affect the rate of hydrolysis of the chimeric oligonucleotides. Thus, there appears to be a major difference in the kinetic behavior of KF and T4 DNA polymerase with respect to the exonuclease reaction. These results are discussed with respect to their possible biological relevance to DNA replication.

Effect of ribonucleotide substitution on nucleic acid bulge recognition by neocarzinostatin

Bulged RNA structures are not as good substrates for cleavage by the enediyne antibiotic neocarzinostatin chromophore in the general base-catalyzed reaction as are DNA bulges. In an effort to determine why this is so, we have systematically substituted ribonucleotide residues in a DNA bulged structure (CCGATGCG·CGCAG T TCGG) (cleaved residue is underlined) known to be an excellent substrate. It was found that ribonucleotide substitution at the bulge target site, as well as at other regions involving duplex formation had a small effect on the cleavage reaction, unless either of the two strands was entirely of the ribo form. By contrast, changing the A·T base pair on the 5′ side of the target nucleotide ( T residue) to ribo A·U resulted in an 87% decrease in cleavage; in fact, conversion of the A alone to the ribo form caused a 68% loss in cleavage. This result can be understood from the recent solution structure of the complex formed between an analogue of the drug radical species and a bulged DNA (Stassinopoulos, A.; Ji, J.; Gao, X.; Goldberg, I.H. Science 1996, 272, 1943), since the 2′ hydroxyl group of the ribo A would be expected to clash sterically with the 7″- O-methyl moiety of the drug. Additional studies on substrate bulge-dependent drug product formation and protection against spontaneous drug degradation support the cleavage experiments, and imply that bulge-specific drug binding is required for efficient cleavage.

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome.

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.

Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [gamma-32P]ATP revealed that 7% of the DNA fragments were covalently linked with mono- to octaribonucleotide primers at their 5' ends. Octaribonucleotides, the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [alpha-32P]GTP, indicating that they were the intact primer RNA with tri- (or di-) phosphate termini, and the shorter ribooligomers were degradation intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were rich in A.

Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [gamma-32P]ATP revealed that 7% of the DNA fragments were covalently linked with mono- to octaribonucleotide primers at their 5' ends. Octaribonucleotides, the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [alpha-32P]GTP, indicating that they were the intact primer RNA with tri- (or di-) phosphate termini, and the shorter ribooligomers were degradation intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were rich in A.

RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro.

A 190-base-pair DNA-RNA hybrid containing the Moloney murine leukemia virus origin of plus-strand DNA synthesis was constructed and used as a source of template-primer for the reverse transcriptase in vitro. Synthesis was shown to initiate precisely at the known plus-strand origin. The observation that some of the origin fragments retained ribonucleotide residues on their 5' ends suggests that the primer for chain initiation is an RNA molecule left behind by RNase H during the degradation of the RNA moiety of the DNA-RNA hybrid. If the RNase H is responsible for creating the correct primer terminus, then it must possess a specific endonucleolytic activity capable of recognizing the sequence in the RNA where plus strands are initiated. The 16-base RNase A-resistant fragment which spans the plus-strand origin can also serve as a source of the specific plus-strand primer RNA. Evidence is presented that some of the plus-strand origin fragments synthesized in the endogenous reaction contain 5' ribonucleotides, suggesting that specific RNA primers for plus-strand initiation may be generated during reverse transcription in vivo as well.

Initiator RNA of nascent DNA from animal cells

Nascent DNA synthesized by intact cells has been examined for the presence of RNA that may function as a primer in the discontinuous synthesis of DNA. A low molecular weight fraction that contains nascent DNA was isolated from a human lymphoblastoid cell line in logarithmic growth. After labeling the 5′ ends with bacteriophage T4 polynucleotide kinase and [γ- 32P]ATP, and digestion of the DNA with DNAase, a DNAase-resistant oligonucleotide was isolated. This fragment consisted of approximately 9 ribonucleotide residues, with 5′ terminal purines ( A G = 3·5 1 ), plus one to three 3′ terminal deoxynucleotides resulting from incomplete removal by DNAase. Approximately 10% of short nascent DNA chains contained the nonanucleotide molecule. An additional 20% of the nascent DNA contained ribooligomers shorter than 9 residues, with 5′ termini substantially increased in pyrimidines, which may result from degradation of the nonanucleotide. These results extend previous studies that demonstrated a similar ribooligonucleotide present at the 5′ end of most or all short nascent DNA chains synthesized in broken cell systems. Together with the results obtained by Reichard and co-workers (Reichard et al., 1974) with polyoma virus, the data support a mechanism by which a short initiator RNA serves as primer for discontinuously synthesized DNA in animal cells.

The use of primed synthesis by DNA polymerase I to study an intercistronic sequence of phiX-174 DNA.

A decadeoxynucleotide complementary to ten nucleotides in the major ribosome-protected fragment of phiX-174 plus-strand DNA has been chemically synthesized and used as a primer for DNA polymerase I on phiX-174 plus-strand DNA as template. The sequence of the first 40 nucleotides incorporated onto the decadeoxynucleotide has been determined. This sequence extends further the sequence of the intercistronic region preceding gene G and shows the presence of another termination codon. The sequence was determined by using manganese as the activating cation for DNA polymerase I which allows ribonucleotides to be incorporated as well as deoxyribonucleotides. The ribo-substituted product was then cleaved specifically at the ribonucleotide residues to generate a series of overlapping ribo-terminated fragments whose sequences were sufficient to determine the complete sequence of the first 40 nucleotides. No evidence for misincorporation by DNA polymerase I in the presence of manganese was detected.

Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase

A method is described for the determination of nucleotide sequences in DNA by using specific oligonucleotides as primers for copying specific regions by DNA polymerase. The method was applied to bacteriophage f1 DNA using the synthetic octanucleotide A-C-C-A-T-C-C-A as primer and a sequence (sequence A) of 81 nueleotides was determined. Synthesis was carried out in the presence of manganese and with one of the deoxyribotriphosphates (dCTP or dGTP) replaced by the corresponding ribotriphosphate so that mixed oligonucleotides were found which could be specifically split at the ribonucleotide residues by the appropriate ribonuclease or by alkali. The relative order of the digestion products was determined by fractionating the undigested oligonucleotides according to size on a two-dimensional system and digesting the isolated products. In the presence of rGTP the octanucleotide appeared to prime at a second site giving rise to a second sequence (B) besides sequence A. The complementary sequence to sequence A, which corresponds to the plus strand of f1 DNA and to the messenger RNA, contains five nonsense codons, four of which are in the same phase, and two possible initiation codons. It also contains a repetitive sequence which suggests its evolutionary origin by duplication.

Proofreading Function of Deoxyribonucleic Acid Polymerase I from Escherichia coli: NATURE OF EXCISION OF RIBONUCLEOTIDES FROM THE 3′ TERMINI OF OLIGODEOXYNUCLEOTIDE PRIMERS

Abstract Oligothymidylic acid containing ribouridylic or riboadenylic acid residues at the 3' terminus was tested as primer for Escherichia coli DNA polymerase I and its large fragment in the presence of polydeoxyadenylic acid as template. Under conditions of synthesis, i.e. in the presence of deoxythymidinetriphosphate and Mg2+, ribouridylic acid residues are preserved during the extension of the primer in contrast to riboadenylic acid residues, which are excised by the 3' → 5' exonuclease activity. Thus, the error corrective function of DNA polymerase only discriminates mismatched from matched bases at the 3' terminus of the primer. Ribonucleotide residues are accepted as long as they allow classical base pairing with the template. In the absence of deoxythymidinetriphosphate, the 3' → 5' exonuclease activity is capable of cleaving both riboadenylic and ribouridylic acid residues from the 3' end of the primer.