The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.RNA degradation ͉ RNA processing ͉ x-ray crystallography ͉ RNase D T he RNA exosome participates in a wide range of reactions, including processing and degradation of tRNA and rRNA as well as degradation of both nuclear and cytoplasmic RNA polymerase II-derived transcripts (1-6). The core eukaryotic exosome is present in both the cytoplasm and nucleus and consists of 10 proteins, with at least 7 harboring proven or predicted 3Ј-to-5Ј exonuclease activity (one RNase II and six RNase PH type) (7-10). In the yeast nucleus, the complex is distinguished by three additional proteins, the RNase D-type enzyme Rrp6p (for ribosomal RNA processing), the DEAD-box RNA helicase Mtr4p, and the less well characterized protein Rrp47p (7, 11). Exosomes are found in both eukaryotes and archaea, and, recently, several crystal structures of archaeal subcomplexes were reported (12)(13)(14). The center of the archaeal exosome consists of three Rrp41 and three Rrp42 proteins forming an overall donut-shaped heterohexameric structure. Rrp41 and -42 are each similar to three proteins in the eukaryotic complex, which consists of six different proteins forming the ''donut' ' (12, 14). This ring-like structure is able to bind additional proteins, forming a ''cap'' suggested to constrict and probably regulate the entry of RNA into the central cavity containing the phosphorolytic active sites (12).The nuclear exosome is essential for maturation of eukaryotic ribosomal RNAs (25S, 18S, and 5.8S), which are synthesized as a single transcript (for a review, see ref. 15). Processing is initiated by endonucleolytic cleavage of the external transcribed spacers (ETSs), which are subsequently degraded by Rrp6p (16). This protein is also required for trimming of the two internal transcribed spacers 1 and 2 during maturation to produce the mature rRNAs, and a 30-nt 3Ј-end extended form of 5.8S rRNA appears in ⌬rrp6 cells (17). Similarly, many small nucleolar RNAs (snoRNAs) depend on the exosome during their maturation (18,19), and deletion of Rrp6p in yeast also leads to accumulation of extended forms of both polycistronic snoRNAs (18) and the independently transcribed snoRNAs, such as snR33 and snR40 (20).Rrp6p is homol...
Temperature is of major importance in most branches of science and technology as well as in everyday life, and with the miniaturization of electronic devices and the increasing ability to make research into small-scale systems, a specific need for very small thermostats and thermometers has been created. Here we describe how DNA molecules can be used as nanoscale sensors to meet these requirements. We illustrate how the hybridization kinetics between bases in DNA molecules combined with conformational changes of the DNA backbone can be exploited in the construction of simple but versatile temperature switches and thermometers, which can be built into electronic systems. DNA based sensors are at the same time applicable as ion detectors to monitor the chemical environment of a specific system.
Deadenylation is the first and probably also rate-limiting step of controlled mRNA decay in eukaryotes and therefore central for the overall rate of gene expression. In yeast, the process is maintained by the mega-Dalton Ccr4-Not complex, of which both the Ccr4p and Pop2p subunits are 3′–5′ exonucleases potentially responsible for the deadenylation reaction. Here, we present the crystal structure of the Pop2p subunit from Schizosaccharomyces pombe determined to 1.4 Å resolution and show that the enzyme is a competent ribonuclease with a tunable specificity towards poly-A. In contrast to S. cerevisiae Pop2p, the S. pombe enzyme contains a fully conserved DEDDh active site, and the high resolution allows for a detailed analysis of its configuration, including divalent metal ion binding. Functional data further indicates that the identity of the ions in the active site can modulate both activity and specificity of the enzyme, and finally structural superposition of single nucleotides and poly-A oligonucleotides provide insight into the catalytic cycle of the protein.
In eukaryotic organisms, initiation of mRNA turnover is controlled by progressive shortening of the poly-A tail, a process involving the mega-Dalton Ccr4-Not complex and its two associated 39-59 exonucleases, Ccr4p and Pop2p (Caf1p). RNA degradation by the 39-59 DEDDh exonuclease, Pop2p, is governed by the classical two metal ion mechanism traditionally assumed to be dependent on Mg 2+ ions bound in the active site. Here, we show biochemically and structurally that fission yeast (Schizosaccharomyces pombe) Pop2p prefers Mn 2+ and Zn 2+ over Mg 2+ at the concentrations of the ions found inside cells and that the identity of the ions in the active site affects the activity of the enzyme. Ion replacement experiments further suggest that mRNA deadenylation could be subtly regulated by local Zn 2+ levels in the cell. Finally, we use site-directed mutagenesis to propose a mechanistic model for the basis of the preference for poly-A sequences exhibited by the Pop2p-type deadenylases as well as their distributive enzymatic behavior.
Although centromere function has been conserved through evolution, apparently no interspecies consensus DNA sequence exists. Instead, centromere DNA may be interconnected through the formation of certain DNA structures creating topological binding sites for centromeric proteins. DNA topoisomerase II is a protein, which is located at centromeres, and enzymatic topoisomerase II activity correlates with centromere activity in human cells. It is therefore possible that topoisomerase II recognizes and interacts with the alpha satellite DNA of human centromeres through an interaction with potential DNA structures formed solely at active centromeres. In the present study, human topoisomerase IIα-mediated cleavage at centromeric DNA sequences was examined in vitro. The investigation has revealed that the enzyme recognizes and cleaves a specific hairpin structure formed by alpha satellite DNA. The topoisomerase introduces a single-stranded break at the hairpin loop in a reaction, where DNA ligation is partly uncoupled from the cleavage reaction. A mutational analysis has revealed, which features of the hairpin are required for topoisomerease IIα-mediated cleavage. Based on this a model is discussed, where topoisomerase II interacts with two hairpins as a mediator of centromere cohesion.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.