Archaeal ß-CASP ribonucleases of the aCPSF1 family are orthologs of the eukaryal CPSF-73 factor.

Bacterial RNase J and eukaryal cleavage and polyadenylation specificity factor (CPSF-73) are members of the ß-CASP family of ribonucleases involved in mRNA processing and degradation. Here we report an in-depth phylogenomic analysis that delineates aRNase J and archaeal CPSF (aCPSF) as distinct orthologous groups and establishes their repartition in 110 archaeal genomes. The aCPSF1 subgroup, which has been inherited vertically and is strictly conserved, is characterized by an N-terminal extension with two K homology (KH) domains and a C-terminal motif involved in dimerization of the holoenzyme. Pab-aCPSF1 (Pyrococcus abyssi homolog) has an endoribonucleolytic activity that preferentially cleaves at single-stranded CA dinucleotides and a 5'-3' exoribonucleolytic activity that acts on 5' monophosphate substrates. These activities are the same as described for the eukaryotic cleavage and polyadenylation factor, CPSF-73, when engaged in the CPSF complex. The N-terminal KH domains are important for endoribonucleolytic cleavage at certain specific sites and the formation of stable high molecular weight ribonucleoprotein complexes. Dimerization of Pab-aCPSF is important for exoribonucleolytic activity and RNA binding. Altogether, our results suggest that aCPSF1 performs an essential function and that an enzyme with similar activities was present in the last common ancestor of Archaea and Eukarya.

PROSITE, a protein domain database for functional characterization and annotation.

PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 ( approximately 70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/.

The 20 years of PROSITE.

PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. In this article, we describe the implementation of a new method to assign a status to pattern matches, the new PROSITE web page and a new approach to improve the specificity and sensitivity of PROSITE methods. The latest version of PROSITE (release 20.19 of 11 September 2007) contains 1319 patterns, 745 profiles and 764 ProRules. Over the past 2 years, about 200 domains have been added, and now 53% of UniProtKB/Swiss-Prot entries (release 54.2 of 11 September 2007) have a PROSITE match. PROSITE is available on the web at: http://www.expasy.org/prosite/.

New developments in the InterPro database.

InterPro is an integrated resource for protein families, domains and functional sites, which integrates the following protein signature databases: PROSITE, PRINTS, ProDom, Pfam, SMART, TIGRFAMs, PIRSF, SUPERFAMILY, Gene3D and PANTHER. The latter two new member databases have been integrated since the last publication in this journal. There have been several new developments in InterPro, including an additional reading field, new database links, extensions to the web interface and additional match XML files. InterPro has always provided matches to UniProtKB proteins on the website and in the match XML file on the FTP site. Additional matches to proteins in UniParc (UniProt archive) are now available for download in the new match XML files only. The latest InterPro release (13.0) contains more than 13 000 entries, covering over 78% of all proteins in UniProtKB. The database is available for text- and sequence-based searches via a webserver (http://www.ebi.ac.uk/interpro), and for download by anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro). The InterProScan search tool is now also available via a web service at http://www.ebi.ac.uk/Tools/webservices/WSInterProScan.html.

The PROSITE database.

The PROSITE database consists of a large collection of biologically meaningful signatures that are described as patterns or profiles. Each signature is linked to a documentation that provides useful biological information on the protein family, domain or functional site identified by the signature. The PROSITE database is now complemented by a series of rules that can give more precise information about specific residues. During the last 2 years, the documentation and the ScanProsite web pages were redesigned to add more functionalities. The latest version of PROSITE (release 19.11 of September 27, 2005) contains 1329 patterns and 552 profile entries. Over the past 2 years more than 200 domains have been added, and now 52% of UniProtKB/Swiss-Prot entries (release 48.1 of September 27, 2005) have a cross-reference to a PROSITE entry. The database is accessible at http://www.expasy.org/prosite/.

ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins.

ScanProsite--http://www.expasy.org/tools/scanprosite/--is a new and improved version of the web-based tool for detecting PROSITE signature matches in protein sequences. For a number of PROSITE profiles, the tool now makes use of ProRules--context-dependent annotation templates--to detect functional and structural intra-domain residues. The detection of those features enhances the power of function prediction based on profiles. Both user-defined sequences and sequences from the UniProt Knowledgebase can be matched against custom patterns, or against PROSITE signatures. To improve response times, matches of sequences from UniProtKB against PROSITE signatures are now retrieved from a pre-computed match database. Several output modes are available including simple text views and a rich mode providing an interactive match and feature viewer with a graphical representation of results.

Recent improvements to the PROSITE database.

The PROSITE database consists of a large collection of biologically meaningful signatures that are described as patterns or profiles. Each signature is linked to documentation that provides useful biological information on the protein family, domain or functional site identified by the signature. The PROSITE web page has been redesigned and several tools have been implemented to help the user discover new conserved regions in their own proteins and to visualize domain arrangements. We also introduced the facility to search PDB with a PROSITE entry or a user's pattern and visualize matched positions on 3D structures. The latest version of PROSITE (release 18.17 of November 30, 2003) contains 1676 entries. The database is accessible at http://www.expasy.org/prosite/.

Universal RNA-degrading enzymes in Archaea: Prevalence, activities and functions of ß-CASP ribonucleases.

ß-CASP ribonucleases are widespread in all three domains of life. They catalyse both 5'-3' exoribonucleolytic RNA trimming and/or endoribonucleolytic RNA cleavage using a unique active site coordinated by two zinc ions. These fascinating enzymes have a key role in 3' end processing in Eukarya and in RNA decay and ribosomal RNA maturation in Bacteria. The recent recognition of ß-CASP ribonucleases as major players in Archaea is an important contribution towards identifying RNA-degrading enzymes in the third domain of life. Three ß-CASP orthologous groups, aCPSF1, aCPSF2, aCPSF1b, are closely related to the eukaryal CPSF73 termination factor and one, aRNase J, is ortholog of the bacterial RNase J. The endo- and 5'-3' exoribonucleolytic activities carried by archaeal ß-CASP enzymes are strictly conserved throughout archaeal phylogeny suggesting essential roles in maturation and/or degradation of RNA. The recent progress in understanding the prevalence, activities and functions of archaeal ß-CASP ribonucleases is the focus of this review. The current status of our understanding of RNA processing pathways in Archaea is covered in light of this new knowledge on ß-CASP ribonucleases.

ProRule: a new database containing functional and structural information on PROSITE profiles.

MOTIVATION: Increase the discriminatory power of PROSITE profiles to facilitate function determination and provide biologically relevant information about domains detected by profiles for the annotation of proteins. SUMMARY: We have created a new database, ProRule, which contains additional information about PROSITE profiles. ProRule contains notably the position of structurally and/or functionally critical amino acids, as well as the condition they must fulfill to play their biological role. These supplementary data should help function determination and annotation of the UniProt Swiss-Prot knowledgebase. ProRule also contains information about the domain detected by the profile in the Swiss-Prot line format. Hence, ProRule can be used to make Swiss-Prot annotation more homogeneous and consistent. The format of ProRule can be extended to provide information about combination of domains. AVAILABILITY: ProRule can be accessed through ScanProsite at http://www.expasy.org/tools/scanprosite. A file containing the rules will be made available under the PROSITE copyright conditions on our ftp site (ftp://www.expasy.org/databases/prosite/) by the next PROSITE release.

In vivo analysis of the overlapping functions of DnaK and trigger factor.

Trigger factor (TF) is a ribosome-bound protein that combines catalysis of peptidyl-prolyl isomerization and chaperone-like activities in Escherichia coli. TF was shown to cooperate with the DnaK (Hsp70) chaperone machinery in the folding of newly synthesized proteins, and the double deletion of the corresponding genes (tig and dnaK) exhibited synthetic lethality. We used a detailed genetic approach to characterize various aspects of this functional cooperation in vivo. Surprisingly, we showed that under specific growth conditions, one can delete both dnaK and tig, indicating that bacterial survival can be maintained in the absence of these two major cytosolic chaperones. The strain lacking both DnaK and TF exhibits a very narrow temperature range of growth and a high level of aggregated proteins when compared to either of the single mutants. We found that, in the absence of DnaK, both the N-terminal ribosome-binding domain and the C-terminal domain of unknown function are essential for TF chaperone activity. In contrast, the central PPIase domain is dispensable. Taken together, our data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.