Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.

Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning.

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas surveillance complexes target double-stranded DNA, type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy reconstructions of native type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumblike ß hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nucleotide intervals. The Cmr complex is architecturally similar to the type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.

Crystal structure of the largest subunit of a bacterial RNA-guided immune complex and its role in DNA target binding.

Prokaryotes make use of small RNAs encoded by CRISPR (clustered regularly interspaced short palindromic repeat) loci to provide immunity against bacteriophage or plasmid invasion. In Escherichia coli, the CRISPR-associated complex for antiviral defense (Cascade) utilizes these RNAs to target foreign DNA for destruction. CasA, the largest subunit of Cascade, is essential for its function. Here we report the crystal structure of Thermus thermophilus CasA. The structure is composed of two domains that are arranged in a chair-like conformation with a novel fold forming the larger N-terminal domain. Docking of the crystal structure into cryo-electron microscopy maps reveals two loops in CasA that likely have important functions in DNA target binding. Finally, DNA binding experiments show that CasA is essential for binding of Cascade to DNA target.

A DNA polymerase III holoenzyme-like subassembly from an extreme thermophilic eubacterium.

We have purified a novel DNA polymerase from Thermus thermophilus. This was enabled by use of general gap filling assays to monitor polymerase activity and cross-reactive monoclonal antibodies against the alpha catalytic subunit of E. coli DNA polymerase III holoenzyme to distinguish a novel polymerase from the well characterized DNA polymerase I-like Thermus thermophilus DNA polymerase. Two proteins migrating with the polymerase after three chromatographic steps were isolated and subjected to partial amino acid sequencing. The amino termini of both were homologous to the two products of the E. coli dnaX gene, the gamma and tau subunits of the DNA polymerase III holoenzyme. Using this information and sequences conserved among dnaX-like genes, we isolated a gene fragment by PCR and used it as a probe to isolate the full length Thermus thermophilus dnaX gene. The deduced amino acid sequence is highly homologous to the DnaX proteins of other bacteria. Examination of the sequence permitted identification of a frameshift site similar to the one used in E. coli to direct the synthesis of the shorter gamma DnaX-gene product. Based on this information, we conclude that a conventional replicase exists in extreme thermophilic eubacteria. The general biological and practical technological implications of this finding are discussed.

Differential domain accessibility to monoclonal antibodies in three different morphological assemblies built up by the S-layer protein of Thermus thermophilus HB8.

A collection of 27 monoclonal antibodies (MAbs) against the S-layer protein (P100) of Thermus thermophilus HB8 has been obtained. They have been classified according to their ability to recognize S-layer regions expressed in E. coli from plasmids containing different fragments of its coding gene, slpA. The accessibility of the binding sites in hexagonal, trigonal, or tetragonal assemblies of P100 was analyzed by enzyme-linked immunosorbent assays with six of these MAbs and their respective Fab fragments. When packed hexagonally as the native S-layer (S1 assemblies), only a small region located near the amino terminus of the P1OO was accessible. However, when P1OO was assembled into trigonal (pS2 assemblies) or tetragonal (S2 assemblies) arrays, most of the protein domains analyzed were easily detected, thus suggesting that P1OO is assembled in S2 and pS2 in a similar way and that these two arrangements are quite different from the S1 assembly. Relationships between accessibility and sequence predictions are discussed.