Structure formation in sugar containing pectin gels – Influence of tartaric acid content (pH) and cooling rate on the gelation of high-methoxylated pectin

The aim of the study was the application of a recently published method, using structuring parameters calculated from dG′/dt, for the characterisation of the pectin sugar acid gelation process. The influence of cooling rate and pH on structure formation of HM pectin gels containing 65 wt.% sucrose were investigated. The results show that the structure formation process as well as the properties of the final gels strongly depended on both parameters. With increasing cooling rates from 0.5 to 1.0 K/min the initial structuring temperature slightly decreased and the maximum structuring velocity increased. The lower the cooling rates, the firmer and more elastic were the final gels. With increasing acid content (decreasing pH from 2.5–2.0) the initial structuring temperatures were nearly constant. The final gel properties varied visibly but not systematically. Gels with the lowest and highest pH were less elastic and weaker compared to those with medium acid concentrations

Combined Biochemical and Electron Microscopic Analyses Reveal the Architecture of the Mammalian U2 snRNP

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5′ half of U2 small nuclear RNA, whereas SF3a associates with the 3′ portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain

Spliceosomal U snRNP Core Assembly: Sm Proteins Assemble onto an Sm Site RNA Nonanucleotide in a Specific and Thermodynamically Stable Manner

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4–6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2′ hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU…) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy

Structure of yeast U6 snRNPs: Arrangement of Prp24p and the LSm complex as revealed by electron microscopy

Protein components of the U6 snRNP (Prp24p and LSm2–8) are thought to act cooperatively in facilitating the annealing of U6 and U4 snRNAs during U4/U6 di-snRNP formation. To learn more about the spatial arrangement of these proteins in S. cerevisiae U6 snRNPs, we investigated the structure of this particle by electron microscopy. U6 snRNPs, purified by affinity chromatography and gradient centrifugation, and then immediately adsorbed to the carbon film support, revealed an open form in which the Prp24 protein and the ring formed by the LSm proteins were visible as two separate morphological domains, while particles stabilized by chemical cross-linking in solution under mild conditions before binding to the carbon film exhibited a compact form, with the two domains in close proximity to one another. In the open form, individual LSm proteins were located by a novel approach employing C-terminal genetic tagging of the LSm proteins with yECitrine. These studies show the Prp24 protein at defined distances from each subunit of the LSm ring, which in turn suggests that the LSm ring is positioned in a consistent manner on the U6 RNA. Furthermore, in agreement with the EM observations, UV cross-linking revealed U6 RNA in contact with the LSm2 protein at the interface between Prp24p and the LSm ring. Further, LSmp–Prp24p interactions may be restricted to the closed form, which appears to represent the solution structure of the U6 snRNP particle