32 research outputs found
Structure and mechanism of the CMR complex for CRISPR-Mediated antiviral immunity
The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endo-nucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a proto-spacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.Publisher PDFPeer reviewe
Characterisation of proteins involved in CRISPR-mediated antiviral defence in Sulfolobus solfataricus
One of the most surprising realisations to emerge from metagenomics studies
in the early â00s was that the population of viruses and phages in nature is about 10
times larger than the population of prokaryotic organisms. Thus, bacteria and archaea
are under constant pressure to develop resistance methods against a population of
viruses with extremely high turnover and evolution rates, in what has been described
as an evolutionary âarms raceâ. A novel, adaptive and heritable immune system
encoded by prokaryotic genomes is the CRISPR/Cas system. Arrays of clustered
regularly interspersed short palindromic repeats (CRISPR) are able to incorporate viral
or plasmid sequences which are then used to inactivate the corresponding invader
element via an RNA interference mechanism. A number of CRISPR-associated (Cas)
protein families are responsible for the maintenance, expansion and function of the
CRISPR loci. This system can be classified in a number of types and subtypes that
differ widely in their gene composition and mode of action.
This thesis describes the biochemical characteristics of CRISPR-mediated
defense in the crenarchaeon Sulfolobus solfataricus. The process of CRISPR loci
transcription and their subsequent maturation into small guide crRNA units by the
processing endonuclease of the system (Cas6) is investigated. After this step, different
pathways and effector proteins are involved in the recognition and silencing of DNA or
RNA exogenous nucleic acids. This thesis reports the identification and purification of
a native multiprotein complex from S. solfataricus P2, the Cmr complex, a homologue
of which has been found to recognise and cleave RNA targets in P. furiosus. The
recognition and silencing of DNA targets in E. coli has been shown to involve a
multiprotein complex termed CASCADE as well as Cas3, a putative helicase-HD
nuclease. S. solfataricus encodes orthologues for the core proteins of this complex,
and the formation and function of an archaeal CASCADE is investigated in this thesis
Ancestral Reconstructions Decipher Major Adaptations of Ammonia-Oxidizing Archaea upon Radiation into Moderate Terrestrial and Marine Environments
International audienc
Structure and mechanism of the CRISPR associated complex for antiviral defense (CASCADE)
Structure and mechanism of the CRISPR associated complex for antiviral defense (CASCADE)
Genome wide transcriptomic analysis of the soil ammonia oxidizing archaeon Nitrososphaera viennensis upon exposure to copper limitation
International audienc
Ammonia Oxidation by the Arctic Terrestrial Thaumarchaeote Candidatus Nitrosocosmicus arcticus Is Stimulated by Increasing Temperatures
International audienc
Proteomics and comparative genomics of Nitrososphaera viennensis reveal the core genome and adaptations of archaeal ammonia oxidizers
International audienceAmmonia-oxidizing archaea (AOA) are among the most abundant microorganisms and key players in the global nitrogen and carbon cycles. They share a common energy metabolism but represent a heterogeneous group with respect to their environmental distribution and adaptions, growth requirements, and genome contents. We report here the genome and proteome of Nitrososphaera viennensis EN76, the type species of the archaeal class Nitrososphaeria of the phylum Thaumarchaeota encompassing all known AOA. N. viennensis is a soil organism with a 2.52-Mb genome and 3,123 predicted protein-coding genes. Proteomic analysis revealed that nearly 50% of the predicted genes were translated under standard laboratory growth conditions. Comparison with genomes of closely related species of the predominantly terrestrial Nitrososphaerales as well as the more streamlined marine Nitrosopumilales [ Candidatus ( Ca. ) order] and the acidophile â Ca. Nitrosotalea devanaterraâ revealed a core genome of AOA comprising 860 genes, which allowed for the reconstruction of central metabolic pathways common to all known AOA and expressed in the N. viennensis and â Ca . Nitrosopelagicus brevisâ proteomes. Concomitantly, we were able to identify candidate proteins for as yet unidentified crucial steps in central metabolisms. In addition to unraveling aspects of core AOA metabolism, we identified specific metabolic innovations associated with the Nitrososphaerales mediating growth and survival in the soil milieu, including the capacity for biofilm formation, cell surface modifications and cell adhesion, and carbohydrate conversions as well as detoxification of aromatic compounds and drugs
A Dimeric Rep Protein Initiates Replication of a Linear Archaeal Virus Genome: Implications for the Rep Mechanism and Viral Replication
The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5' end of the DNA, releasing a 3' DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed.</p