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

Effect of 6S RNA on the transcription from σ and σ-specific promoters on linear DNA fragments

<p><b>Copyright information:</b></p><p>Taken from "Studies on the function of the riboregulator 6S RNA from : RNA polymerase binding, inhibition of transcription and synthesis of RNA-directed transcripts"</p><p></p><p>Nucleic Acids Research 2007;35(6):1885-1896.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874619.</p><p>© 2007 The Author(s)</p> Products from transcription reactions were separated on denaturing polyacrylamide gels and visualized by autoradiography. Reactions with the or P1 promoters are shown on the left or right side, respectively. The different holoenzymes employed (E70, E38) are indicated above the lanes. For each system the amount of 6S RNA present in the reaction was varied (lanes 1, 6, 11, 16: 0 nM, lane 2, 7, 12, 17: 10 nM, lane 3, 8, 13, 18: 50 nM, lane 4, 9, 14, 19: 100 nM, lane 5, 10, 15, 20: 250 nM). A 260 bp radiolabelled DNA fragment, indicated at the margin, was included as internal standard for quantification. The positions of the run-off transcripts for the (∼124 nt) and P1 (64 nt) promoters are marked. An arrow denotes a product that consistently arises when 6S RNA is incubated with RNA polymerase, even in the absence of any template DNA