RESUMO
High-quality chromosomal DNA is a requirement for many biochemical and molecular biological techniques. To isolate cellular DNA, standard protocols typically lyse cells and separate nucleic acids from other biological molecules using a combination of chemical and physical methods. After a standard chemical-based protocol to isolate chromosomal DNA from Saccharomyces cerevisiae and then treatment with RNase A to degrade RNA, two RNase-resistant bands persisted when analyzed using gel electrophoresis. Interestingly, such resistant bands did not appear in preparations of Escherichia coli bacterial DNA after RNase treatment. Several enzymatic, chemical, and physical methods were employed in an effort to remove the resistant RNAs, including use of multiple RNases and alcohol precipitation, base hydrolysis, and chromatographic methods. These experiments resulted in the development of a new method for isolation of S. cerevisiae chromosomal DNA. This method utilizes selective precipitation of DNA in the presence of a potassium acetate/isopropanol mixture and produces high yields of chromosomal DNA without detectable contaminating RNAs.
Assuntos
Cromossomos/genética , DNA/isolamento & purificação , RNA/metabolismo , Ribonucleases/metabolismo , Saccharomyces cerevisiae/genética , 2-Propanol/química , Precipitação Química , Escherichia coli/genética , Hidrólise , Acetato de Potássio/químicaRESUMO
Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5' splice-site (5'SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28's ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease H/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genéticaRESUMO
The spliceosome is a dynamic macromolecular machine that undergoes a series of conformational rearrangements as it transitions between the several states required for accurate splicing. The transition from the B to Bact is a key part of spliceosome assembly and is defined by the departure of several proteins, including essential U5 component Dib1. Recent structural studies suggest that Dib1 has a role in preventing premature spliceosome activation, as it is positioned adjacent to the U6 snRNA ACAGAGA and the U5 loop I, but its mechanism is unknown. Our data indicate that Dib1 is a robust protein that tolerates incorporation of many mutations, even at positions thought to be key for its folding stability. However, we have identified two temperature-sensitive mutants that stall in vitro splicing prior to the first catalytic step and block assembly at the B complex. In addition, Dib1 readily exchanges in splicing extracts despite being a central component of the U5 snRNP, suggesting that the binding site of Dib1 is flexible. Structural analyses show that the overall conformation of Dib1 and the mutants are not affected by temperature, so the temperature sensitive defects most likely result from altered interactions between Dib1 and other spliceosomal components. Together, these data lead to a new understanding of Dib1's role in the B to Bact transition and provide a model for how dynamic protein-RNA interactions contribute to the correct assembly of a complex molecular machine.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Precursores de RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sítios de Ligação , DNA Helicases/química , Modelos Moleculares , Mutação , Conformação Proteica , Dobramento de Proteína , Splicing de RNA , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , TermodinâmicaRESUMO
Montmorillonite (MMT) nanoclays exist as single and stacked sheet-like structures with large surface areas that can form stable associations with many naturally occurring biomolecules, including nucleic acids. They have been utilized successfully as vehicles for delivery of both drugs and genes into cells. Most previous studies have focused on interactions of MMT with DNA. In the current study, we have investigated the binding of small RNAs similar to those used for RNA interference (RNAi) therapy to two major forms of the clay, Na-MMT and Ca-MMT. Association of both forms of MMT with several double-stranded RNAs (dsRNAs), including 25mers, 54mers and cloverleaf-shaped transfer RNAs, was weak and increased only slightly after addition of Mg2+ ions to the binding reactions. By contrast, ssRNA 25mers and 54mers bound poorly to Na-MMT but interacted strongly with Ca-MMT. The weak binding of ssRNAs to Na-MMT could be strongly enhanced by addition of Mg2+ ions. The strength of MMT-ssRNA interactions was also examined using inorganic anion competition and displacement assays, as well as electrophoretic mobility shift assays (EMSAs). The aggregate results point to a cation-bridging mechanism for binding of ssRNAs, but not dsRNAs, in the presence of divalent metal cations.
Assuntos
Bentonita/química , Nanoestruturas/química , RNA de Cadeia Dupla/química , Sequência de Bases , Magnésio/química , Conformação de Ácido Nucleico , Sódio/químicaRESUMO
A number of small, basic proteins penetrate into the structure of the large subunit of the ribosome. While these proteins presumably aid in the folding of the rRNA, the extent of their contribution to the stability or function of the ribosome is unknown. One of these small, basic proteins is L36, which is highly conserved in Bacteria, but is not present in Archaea or Eucarya. Comparison of ribosome crystal structures shows that the space occupied by L36 in a bacterial ribosome is empty in an archaeal ribosome. To ask what L36 contributes to ribosome stability and function, we have constructed an Escherichia coli strain lacking ribosomal protein L36; cell growth is slowed by 40-50% between 30 degrees C and 42 degrees C. Ribosomes from this deletion strain sediment normally and have a full complement of proteins, other than L36. Chemical protection experiments comparing rRNA from wild-type and L36-deficient ribosomes show the expected increase in reagent accessibility in the immediate vicinity of the L36 binding site, but suggest that a cooperative network of rRNA tertiary interactions has been disrupted along a path extending 60 A deep into the ribosome. These data argue that L36 plays a significant role in organizing 23 S rRNA structure. Perhaps the Archaea and Eucarya have compensated for their lack of L36 by maintaining more stable rRNA tertiary contacts or by adopting alternative protein-RNA interactions elsewhere in the ribosome.
Assuntos
RNA Bacteriano/química , RNA Ribossômico 23S/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Bases , Sítios de Ligação , Escherichia coli , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/genética , Ribossomos/genéticaRESUMO
Use of ribonucleic acid (RNA) interference to regulate protein expression has become an important research topic and gene therapy tool, and therefore, finding suitable vehicles for delivery of small RNAs into cells is of crucial importance. Layered double metal hydroxides such as hydrotalcite (HT) have shown great promise as nonviral vectors for transport of deoxyribose nucleic acid (DNA), proteins, and drugs into cells, but the adsorption of RNAs to these materials has been little explored. In this study, the binding of small RNAs with different lengths and levels of secondary structure to HT nanoparticles has been analyzed and compared to results obtained with small DNAs in concurrent experiments. Initial experiments established the spectrophotometric properties of HT in aqueous solutions and determined that HT particles could be readily sedimented with near 100% efficiencies. Use of RNA+HT cosedimentation experiments as well as electrophoretic mobility shift assays demonstrated strong adsorption of RNA 25mers to HT, with twofold greater binding of single-stranded RNAs relative to double-stranded molecules. Strong affinities were also observed with ssRNA and dsRNA 54mers and with more complex transfer RNA molecules. Competition binding and RNA displacement experiments indicated that RNA-HT associations were strong and were only modestly affected by the presence of high concentrations of inorganic anions.
Assuntos
Adsorção , Hidróxido de Alumínio/análise , Sistemas de Liberação de Medicamentos , Hidróxido de Magnésio/análise , Nanopartículas/química , RNA/metabolismo , Transporte Biológico , RNA/químicaRESUMO
The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. During splicing in vitro, we observed a multitude of generally reversible time- and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3'-splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.
Assuntos
Conformação de Ácido Nucleico , Precursores de RNA/química , Splicing de RNA , RNA Fúngico/química , RNA Mensageiro/química , Transferência Ressonante de Energia de FluorescênciaRESUMO
The spliceosome is a highly dynamic machine requiring multiple RNA-dependent ATPases of the DExD/H-box family. A fundamental unanswered question is how their activities are regulated. Brr2 function is necessary for unwinding the U4/U6 duplex, a step essential for catalytic activation of the spliceosome. Here we show that Brr2-dependent dissociation of U4/U6 snRNAs in vitro is activated by a fragment from the C terminus of the U5 snRNP protein Prp8. In contrast to its helicase-stimulating activity, this fragment inhibits Brr2 U4/U6-dependent ATPase activity. Notably, U4/U6 unwinding activity is not stimulated by fragments carrying alleles of prp8 that in humans confers an autosomal dominant form of retinitis pigmentosa. Because Brr2 activity must be restricted to prevent premature catalytic activation, our results have important implications for fidelity maintenance in the spliceosome.
Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Genes Dominantes , Humanos , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Desnaturação Proteica , RNA Helicases/genética , Splicing de RNA/genética , RNA Fúngico/genética , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Retinose Pigmentar/genética , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genéticaRESUMO
Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2's helicase-like domains and domains 1-3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.
Assuntos
Adenosina Trifosfatases/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Helicases/química , Proteínas de Saccharomyces cerevisiae/química , Spliceossomos/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Humanos , Dados de Sequência Molecular , RNA Helicases/genética , RNA Helicases/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
A conserved, independently folding domain in the large ribosomal subunit consists of 58 nt of rRNA and a single protein, L11. The tertiary structure of an rRNA fragment carrying the Escherichia coli sequence is marginally stable in vitro but can be substantially stabilized by mutations found in other organisms. To distinguish between possible reasons why natural selection has not evolved a more stable rRNA structure in E. coli, mutations affecting the rRNA tertiary structure were assessed for their in vitro effects on rRNA stability and L11 affinity (in the context of an rRNA fragment) or in vivo effects on cell growth rate and L11 content of ribosomes. The rRNA fragment stabilities ranged from -4 to +9 kcal/mol relative to the wild-type sequence. Variants in the range of -4 to +5 kcal/mol had almost no observable effect in vivo, while more destabilizing mutations (>7 kcal/mol) were not tolerated. The data suggest that the in vivo stability of the complex is roughly -6 kcal/mol and that any single tertiary interaction is dispensable for function as long as a minimum stability of the complex is maintained. On the basis of these data, it seems that the evolution of this domain has not been constrained by inherent structural or functional limits on stability. The estimated stability corresponds to only a few ribosomes per bacterial cell dissociated from L11 at any time; thus the selective advantage for any further increase in stability may be so small as to be outweighed by other competing selective pressures.