Optimization of a Ribosomal Structural Domain by Natural Selection

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 (\u3e7 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

Identification of RNase-Resistant RNAs in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Extracts: Separation from Chromosomal DNA by Selective Precipitation

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

Factors Affecting the Association of Single- and Double-Stranded RNAs with Montmorillonite Nanoclays

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

Functional and Biochemical Characterization of Dib1\u27s Role in Pre-Messenger RNA Splicing

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\u27s 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

Impact of Size, Secondary Structure, and Counterions on the Binding of Small Ribonucleic Acids to Layered Double Hydroxide Nanoparticles

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

Conformational Dynamics of Single pre-mRNA Molecules During \u3cem\u3eIn Vitro\u3c/em\u3e Splicing

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

ATP-Dependent Unwinding of U4/U6 snRNAs by the Brr2 Helicase Requires the C Terminus of Prp8

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

ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8.

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