by Alexandra Grabowska
CRISPR (clustered regularly interspaced short palindromic repeats) is a prokaryotic system of acquired immunity. Its mechanism is similar in action to the one of RNAi in eukaryotic organisms and it gained quite a lot of attention in recent years. These adaptive immunity systems are present a great part of already known archaeal and bacterial genomes.
CRISPR loci are built of short direct repeats which are separated by spacers of exogenous origin. During the invasion of phage or a mobile genetic element, invasive nucleic acid can get cleaved by bacterial protein machinery. Part of alien sequence is then incorporated into specific genomic loci and gets transcribed with the rest of CRISPR units, in one long RNA chain. After being cleaved, transcripts are used as guide RNAs to destroy the invasive virus or plasmid. Specificity of this unique immune system relies on the formation of R-loops, which is formed between the RNA-spacer sequence and the complementary target DNA.
Cas, CRISPR-associated proteins, is a cluster of genes adjacent to arrays of repeat-spacer units. Because direct repeats vary in great degree between bacteria and spacers are unique alien sequences, cas genes can be used to determine the relationships between systems and their origin. Systems were divided into three types and later subtypes. In all of them cas1 and cas2 constitute the universal core of system. Cas1 is a metal-dependent DNase of no sequence specificity and is probably involved in the first step of CRISPR mechanism – integration of the new spacer into genome. Despite its presence in all CRISPR loci, the function of Cas2 remains elusive. Using freely available software and databases I tried to investigate this on my own.
A reliable source of information is the work on Cas2 structure from Desulfovibrio vulgaris (DvuCas2), presenting results obtained in crystallographic research. Cas2 in most of bacteria is not a very big protein. DvuCas2 is a homodimer protein built of 102 amino acids. Each protomer contains an N-terminal βαββαβ ferredoxin fold joined with the fifth β-strand and a short helix on the C-terminus. The whole dimer is stabilized by hydrogen bonds.
In studies of Cascade (complex of other CRISPR-associated proteins, not involving Cas2), it was shown that the complex is able to cleave the pre-crRNA transcript in vitro. It means, that Cas2 is not necessary for RNA cleaving or processing in step of CRISPR expression. The study of Beloglazova (2008) showed metal-dependent endonuclease activity of the protein. Homolog from Thermotoga maritima and a few other strains also had RNase activity. Due to these results, the proposed function of Cas2 was selective degradation of phage transcripts or global translation inhibition via mRNA cleavage. Looking for protein motifs using freely available scanner showed no results.
Cas1 and Cas2 are the most conserved proteins in cas cluster. Because of this fact I chose to compare cas2 between chosen strains. In order to investigate some relationship between DvuCas2 and other cas2 genes, I used pairwise alignment for cas2 genes from 6 strains, including Desulfovibrio vulgaris. The greatest similarity was detected for Desulfovibrio vulgaris, Haloarcula marismortui and Thermotoga thermarum. The result is quite plausible. In the recent CRISPR classification systems of these strains (former Tneap-Hmari and Dvulg types) were shown to be related, as they shared a common gene of the BH0338 family. Type found in Aeropyrum pernix (former Apern) also shares this gene, but in this simple alignment there was no closer relationship shown.
CRISPR system is still quite a new discovery in area of bacterial genetics research. Because of potential interesting applies of the system, a lot of work is done to elucidate the mechanism of action and functions of proteins. Cas2 presence and conservation among different types is the evidence on its important role in functioning of CRISPR. Up till now, it remains elusive and mysterious.
Bibliography:
N. Beloglazova et alteres, A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J. Biol. Chem 2008 Jul 18;283(29):20361-71.
S. J. J. Brouns et alteres, Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes.
Science 15 August 2008: 321 (5891), 960-964.
P. Samai et alteres (2010), Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. Acta Crystallographica Section F, 66: 1552–1556.
Source of Cas2 structure: RCSB Protein Data Bank
PJ.L. Moreland, A.Gramada, O.V. Buzko, Q. Zhang and P.E. Bourne 2005 The Molecular Biology Toolkit (mbt): A Modular Platform for Developing Molecular Visualization Applications. BMC Bioinformatics, 6:2 1
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interesting work ! nicely done and nice conclusions !
vote for pedro