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.

The E. coli Anti-Sigma Factor Rsd: Studies on the Specificity and Regulation of Its Expression

Background: Among the seven different sigma factors in E. coli s 70 has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting s 70-dependent transcription at the onset of stationary growth. Although binding of Rsd to s 70 has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown. Methodology/Principal Findings: We have performed studies to unravel the function and regulation of Rsd expression in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with s 70, with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with s 38, the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and s 38-ors 70-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of s 70-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most notably, the dam-dependent methylation of a cluster of GATC sites turned ou

6S RNA-dependent inhibition of RNA polymerase is released by RNA-dependent synthesis of small<i>de novo</i>products

Abstract6S RNA fromEscherichia coliis known to bind to RNA polymerase, preventing interaction with many promoters during stationary growth. The resulting repression is released under conditions of nutritional upshift, when the growth situation improves. 6S RNA, which binds to the active site of RNA polymerase, has the particularly interesting feature to act as a template, causing the transcription of definedde novoRNAs (dnRNA) that are complementary to a specific sequence region of the 6S RNA. We analyzed the conditions of dnRNA synthesis and determined their effect on the 6S RNA-mediated inhibition of RNA polymerasein vitroandin vivo. Upon nutritional upshift the RNA polymerase/6S RNA complex induces the rapid synthesis of dnRNAs, which form stable hybrids with the 6S RNA template. The resulting structural change destabilizes the inactivated RNA polymerase complex, causing σ subunit release. Both dnRNA and 6S RNA are rapidly degraded after complex disintegration. Experiments using the transcriptional inhibitor rifampicin demonstrate that active transcription is required for the disintegration of the RNA polymerase/6S RNA complex. Our results support the conclusion that 6S RNA not only inhibits transcription during stationary growth but also enables cells to resume rapid growth after starvation and help to escape from stationary phase.</jats:p