Binding and cleavage of CRISPR RNA by Cas6.

The CRISPR-Cas system provides many prokaryotes with acquired resistance to viruses and other mobile genetic elements. The core components of this defense system are small, host-encoded prokaryotic silencing (psi)RNAs and Cas (CRISPR-associated) proteins. Invader-derived sequences within the psiRNAs guide Cas effector proteins to recognize and silence invader nucleic acids. Critical for CRISPR-Cas defense is processing of the psiRNAs from the primary transcripts of the host CRISPR (clustered regularly interspaced short palindromic repeat) locus. Cas6, a previously identified endoribonuclease present in a wide range of prokaryotes with the CRISPR-Cas system, binds and cleaves within the repeat sequences that separate the individual invader targeting elements in the CRISPR locus transcript. In the present study, we investigated several key aspects of the mechanism of function of Cas6 in psiRNA biogenesis. RNA footprinting reveals that Pyrococcus furiosus Cas6 binds to a 7-nt (nucleotide) sequence near the 5' end of the CRISPR RNA repeat sequence, 14 nt upstream of the Cas6 cleavage site. In addition, analysis of the cleavage activity of P. furiosus Cas6 proteins with mutations at conserved residues suggests that a triad comprised of Tyr31, His46, and Lys52 plays a critical role in catalysis, consistent with a possible general acid-base RNA cleavage mechanism for Cas6. Finally, we show that P. furiosus Cas6 remains stably associated with its cleavage products, suggesting additional roles for Cas6 in psiRNA biogenesis.

Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella.

The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.

Generation of multicolored, prestained molecular weight markers for gel electrophoresis.

Molecular weight marker proteins are routinely used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis to estimate the relative molecular mass of specific proteins within a sample. This report describes a simple procedure for the generation of multicolored molecular weight proteins using a variety of Remazol-reactive textile dyes. These multicolored proteins provide a set of unambiguous markers for gel electrophoresis. Furthermore, the colored markers can be used in conjunction with Western blotting techniques to provide a visual display of marker proteins on the transfer membrane.