Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis

Nowotny, Marcin; Gaidamakov, Sergei; Crouch, Robert J.; Yang, Wei

doi:10.1016/j.cell.2005.04.024

Cited by 564 publications

(851 citation statements)

References 43 publications

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“…Furthermore, cleavage occurred only with guide RNAs bearing sequence complementarity to the substrate strand. We next tested substrate cleavage kinetics in the presence of different divalent metal ions, which are required for Ago activity (9,29), to explore whether divalent metal ion identity contributes to the discrimination between 5′ hydroxyl and 5′ phosphate guide RNA termini (30) (Fig. S2A).…”

Section: Resultsmentioning

confidence: 99%

A bacterial Argonaute with noncanonical guide RNA specificity

Kaya

Doxzen

Knoll

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

132

232

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Eukaryotic Argonaute proteins induce gene silencing by small RNAguided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPRcas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide.Argonaute | small noncoding RNA | RNA interference A rgonaute (Ago) proteins bind small RNA or DNA guides, which provide base-pairing specificity for recognition and cleavage of complementary nucleic acid targets. Members of this protein family are present in all three domains of life (1). In eukaryotes, Argonautes are the key effectors of RNA interference (RNAi) pathways that regulate posttranscriptional gene expression (2-4). However, the role of Argonaute proteins in bacteria and archaea, which lack RNAi pathways, remains poorly understood (5).Recent studies suggested that DNA-guided bacterial and archaeal Argonaute proteins are directly involved in host defense by cleaving foreign DNA elements, such as DNA viruses and plasmids (6, 7). In addition, a catalytically inactive Argonaute protein in Rhodobacter sphaeroides (RsAgo) was demonstrated to use RNA guides and possibly recruits an associated nuclease for subsequent target cleavage (8). Despite these divergent modes of action, bacterial and archaeal Argonaute proteins adopt a highly conserved bilobed architecture. Herein, an N-terminal and a PIWI-ArgonauteZwille (PAZ) domain constitute one lobe, whereas the other lobe consists of the middle (MID) domain and the catalytic RNase H-like P element-induced wimpy testis (PIWI) domain (9-15). Molecular structures of a eukaryotic Argonaute MID domain and an Archaeoglobus fulgidus Piwi (AfPiwi) enzyme bound to a guide RNA showed the importance of the 5′-terminal base identity, as well as the 5′ phosphate in guide strand binding, to Ago (10,[13][14][15][16][17]. Notably, recognition of the 5′ end of the guide in the MID domain and guide strand preorganization for target interaction are conserved across the entire Argonaute superfamily (1).The nucleic acid-guided binding and cleavage activities of Argonaute proteins are reminiscent of the activities of RNA-guided proteins within CRISPR-Cas systems [cluste...

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Section: Resultsmentioning

confidence: 99%

A bacterial Argonaute with noncanonical guide RNA specificity

Kaya

Doxzen

Knoll

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

132

232

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“…This result casts some doubt on the hypothesis of a strong metal-binding site on the enzyme. It is possible that an efficient Mn 2ϩ coordination may occur only in the ternary complex with RNA, as in RNaseP (17) and RNaseH (18).…”

Section: Resultsmentioning

confidence: 99%

The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication

Renzi

Caffarelli

Laneve

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

107

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Small nucleolar RNAs (snoRNAs) play a key role in eukaryotic ribosome biogenesis. In most cases, snoRNAs are encoded in introns and are released through the splicing reaction. Some snoRNAs are, instead, produced by an alternative pathway consisting of endonucleolytic processing of pre-mRNA. XendoU, the endoribonuclease responsible for this activity, is a U-specific, metaldependent enzyme that releases products with 2 -3 cyclic phosphate termini. XendoU is broadly conserved among eukaryotes, and it is a genetic marker of nidoviruses, including the severe acute respiratory syndrome coronavirus, where it is essential for replication and transcription. We have determined by crystallography the structure of XendoU that, by refined search methodologies, appears to display a unique fold. Based on sequence conservation, mutagenesis, and docking simulations, we have identified the active site. The conserved structural determinants of this site may provide a framework for attempting to design antiviral drugs to interfere with the infectious nidovirus life cycle.crystallography ͉ NendoU ͉ protein structure ͉ RNase family S mall nucleolar RNAs (snoRNAs), required for processing and modification of rRNA, are either independently transcribed or encoded in introns. The generation of most intronencoded snoRNAs relies on the splicing reaction and leads to equimolar accumulation of spliced mRNA and snoRNA (1). Some snoRNAs are, instead, produced by a splicing-independent pathway: Endonucleolytic cleavages of the pre-mRNA release a pre-snoRNA that is converted to the mature form by exonucleolytic trimming (2, 3). In yeast, Rnt1p was shown to be the endonuclease responsible for the excision of intron-encoded snoRNAs and to be activated by the interaction with snoRNP factors assembled on the nascent transcript (3). Among higher eukaryotes, the endoribonuclease from Xenopus laevis, called XendoU, was shown to be responsible for processing the intronencoded U16 and U86 snoRNAs (2, 4-6). XendoU cuts the RNA substrate at the level of short single-stranded uridine stretches. XendoU is unique among known endoribonucleases, because it generates products with 2Ј-3Ј cyclic phosphate and 5Ј OH termini and requires Mn 2ϩ as an essential cofactor. XendoU is broadly conserved among metazoans (HomoloGene: 48394), even if the function of the homologous proteins is still hypothetical, as in the case of the human homolog (hpp11), described as a putative serine protease (7). Notably, a protein with sequence similarity to XendoU has been recently characterized in ssRNA(ϩ) viruses of the Nidovirales order, including the coronavirus (CoV) responsible for the severe acute respiratory syndrome (SARS) (8). Studies aimed at characterizing the genome-proteome of SARS-CoV led to the isolation of a homolog of XendoU called NendoU. NendoU is a component of the replicase-transcriptase complex and exerts a critical role in virus replication and transcription (9). Interestingly, NendoU was found to cleave RNA at the level of uridines, releasing 2Ј-3Ј cyclic phosph...

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“…This position makes specific contacts with RT residues T473 and Q475 located within the RNase H primer grip 19,22 . These residues are conserved between RNases H found in viruses, bacteria and humans 22,31,32 . Replacement of these residues with alanine in HIV-1 RT decreases the DNA synthesis rate and inhibits virus infectivity 33 .…”

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confidence: 99%

Dynamic binding orientations direct activity of HIV reverse transcriptase

Abbondanzieri¹,

Bokinsky²,

Rausch

et al. 2008

Nature

171

216

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The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.Virtually all RNA-processing and DNA-processing enzymes show selectivity for backbone compositions or base sequences of their nucleic-acid substrates. This substrate selectivity is especially crucial for the HIV-1 reverse transcriptase (RT), which binds and discriminates between a variety of nucleic-acid duplexes for distinct catalytic functions 1,2 . RT is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains 3,4 , catalysing a complex, multi-step reaction to convert the single-stranded RNA genome into double-stranded DNA 1,2 . First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA 11-13 , thereby creating the double-stranded DNA viral genome. Specific cleavage by RNase H then removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral DNA into the host chromosome 14 . Inappropriate initiation of synthesis of the plus-strand DNA at other RNA segments prevents integration 2,15 . RT must therefore obey the following primer-selection rules: first, DNA primers readily engage the polymerase activity of RT; second, generic RNA primers are not efficiently extended by RT but readily engage the RNase H activity of RT when annealed with DNA; third, the PPT RNA ca...

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Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis

Cited by 564 publications

References 43 publications

A bacterial Argonaute with noncanonical guide RNA specificity

A bacterial Argonaute with noncanonical guide RNA specificity

The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication

Dynamic binding orientations direct activity of HIV reverse transcriptase

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