Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

La Real Casa dei Matti di Palermo: storia di un'innovazione terapeutica tra architettura e letteratura

La storia di questa struttura psichiatrica rappresenta uno straordinario esempio di come innovazione medica, letteratura e architettura si intreccino formando un\u2019unica trama storica che merita di essere letta e tenuta in considerazion

The Hyperthermophilic Archaeon Sulfolobus: From Exploration to Exploitation

In the early 1970s, Sulfolobus was first isolated by Thomas Brock and co-workers from sulfur-rich acidic hot springs in Yellowstone National Park. Sulfolobus became one of the model organisms of Archaea in general, and of Crenarchaea in particular. Many of its unusual physiological characteristics have been investigated, and several of its thermostable enzymes have been studied in considerable detail. For fundamental reasons, and because of its potential for industrial applications, Sulfolobus has been selected for a genome sequence project. The recent completion of the Sulfolobus solfataricus genome has set the stage for a series of postgenome research lines that will be reviewed here. Comparative genomics aims to unravel the cell’s metabolic potential (enzymes, pathways, and regulation) and compare it to other organisms. This in silico analysis of Sulfolobus has revealed several unique metabolic features and confirmed that the major control processes (transcription, translation, and replication) resemble eukaryal much more than bacterial equivalents. Currently, several research groups use a functional genomics approach—a genomebased, high-throughput analysis of the complete Sulfolobus cell—at the level of RNA (transcriptomics), protein (proteomics), and metabolic intermediates and products (metabolomics). Apart from analyzing the cell’s response to different cultivation conditions, the comparison of different genotypes (wild type and mutants) would be very interesting for both basic science and applied reasons. Much effort has recently been put into the establishment of tools that allow genetic engineering of Sulfolobus. The future challenge is to integrate available knowledge in order to understand the relevant mechanisms that enable Sulfolobus to thrive in its extreme habitats. Moreover, such a Systems Biology approach is essential as a basis for the directed engineering and industrial exploitation of this unique microorganism

Identification of a Novel α-Galactosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable α-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for α-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable α-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea

Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis

In the last decade, an increasing number of sequenced archaeal genomes have become available, opening up the possibility for functional genomic analyses. Here, we reconstructed the central carbon metabolism in the hyperthermophilic crenarchaeon Sulfolobus solfataricus (glycolysis, gluconeogenesis and tricarboxylic acid cycle) on the basis of genomic, proteomic, transcriptomic and biochemical data. A 2-DE reference map of S. solfataricus grown on glucose, consisting of 325 unique ORFs in 255 protein spots, was created to facilitate this study. The map was then used for a differential expression study based on 15N metabolic labelling (yeast extract + tryptone-grown cells (YT) vs. glucose-grown cells (G)). In addition, the expression ratio of the genes involved in carbon metabolism was studied using DNA microarrays. Surprisingly, only 3 and 14% of the genes and proteins, respectively, involved in central carbon metabolism showed a greater than two-fold change in expression level. All results are discussed in the light of the current understanding of central carbon metabolism in S. solfataricus and will help to obtain a system-wide understanding of this organism.</p