No description available.

No description available.

Most eukaryotic pre-mRNAs undergo 3′-end cleavage and polyadenylation prior to their export from the nucleus. A large number of proteins in several complexes participate in this 3′-end processing, including cleavage and polyadenylation specificity factor (CPSF) in mammals. The CPSF can be further divided into two sub-complexes: mPSF (mammalian polyadenylation specificity factor) which recognizes the AAUAAA polyadenylation signal (PAS) in the pre- mRNA, and mCF (mammalian cleavage factor) which cleaves the RNA. mPSF consists of CPSF160, CPSF30, WDR33, and hFip1. This thesis shows that AAUAAA PAS is recognized with ∼3 nM affinity by the CPSF160–WDR33–CPSF30 ternary complex, while the proteins alone or the binary complexes do not bind the PAS with high affinity. Furthermore, it is shown that mutations of residues in CPSF30 that have van der Waals interactions with the bases of the PAS lead to a sharp reduction in the affinity. Finally, variations of the AAUAAA or removing the bases downstream also reduce the binding significantly. This thesis goes on to characterize the structure of the CPSF30—hFip1 complex, which was not observed in the previous EM structures of the mPSF. It was known that CPSF30 ZF4–ZF5 recruits the hFip1 subunit of CPSF, although the details of this interaction have not been characterized. Here we report the crystal structure of human CPSF30 ZF4–ZF5 in complex with residues 161–200 of hFip1 at 1.9 Å. Unexpectedly, the structure reveals one hFip1 molecule binding to each ZF4 and ZF5, with a conserved mode of interaction. Mutagenesis studies confirm that the CPSF30–hFip1 complex has 1:2 stoichiometry in vitro. Mutation of each binding site in CPSF30 still allows one copy of hFip1 to bind, while mutation of both sites abrogates binding. Our fluorescence polarization binding assays show that ZF4 has higher affinity for hFip1, with a Kd of 1.8 nM. We also demonstrate that two copies of the catalytic module of poly(A) polymerase (PAP) are recruited by the CPSF30–hFip1 complex in vitro, and both hFip1 binding sites in CPSF30 can support polyadenylation.

To study the mRNA found in sheep omasal epithelium encoding for a peptide transport protein(s), a 446-bp cDNA fragment was cloned from sheep omasal epithelium RNA. The predicted amino acid sequence of this fragment was 85.8, 90.5, and 90.5 percent identical to rabbit, human, and rat PepT1, respectively. The fragment was radiolabeled for use as a probe to study the distribution of the mRNA in various tissues. Total RNA was extracted and mRNA was isolated from the epithelium of gastrointestinal segments and other tissues as indicated. Northern blot analysis was conducted using the radiolabeled probe. In sheep (5) and lactating Holstein cows (3), hybridization was observed with mRNA from the omasum, rumen, duodenum, jejunum, and ileum. The estimated size of mRNA was 2.8 kb. No hybridization was observed with mRNA from the abomasum, cecum, colon, liver, kidney, and semitendinosus and longissimus muscles of either species or the mammary gland of the dairy cows. In pigs (6), the probe hybridized with mRNA from the duodenum, jejunum, and ileum. There was no hybridization with mRNA from the stomach, large intestine, liver, kidney, and semitendinosus and longissimus muscles. Two bands, 3.5 and 2.9 kb were observed with northern blot analysis, indicating two RNA transcripts that may result from alternative mRNA processing. In both Leghorns (15) and broilers (20), the strongest hybridization was found in the duodenum while the jejunum and ileum showed faint bands. The size of mRNA in chickens was 1.9 kb. Other tissues, including the crop, proventriculus, gizzard, ceca, liver, kidney, and muscles showed no hybridization to the probe. In conclusion, mRNA for a peptide transport protein(s) is present in the small intestine of all animals examined and the omasal and ruminal epithelium of sheep and dairy cows. The size of the mRNA varied among species. / Master of Science

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

The purposes of this study was to demonstrate the induction of alpha interferon mRNA in Sendai virus-induced Namalava cells, to follow the level of alpha interferon mRNA synthesis at the transcriptional level, and to determine whether the Namalava cell line expresses the c-myc oncogene and to what degree. The amount of c-myc message deteted in Namalva cell RNA was about one-tenth that of Daudi cell RNA, whereas no difference in the amount of the c-Ha-ras message was observed between the two cell lines.

Pre-mRNA molecules in humans contain mostly short internal exons flanked by long introns. To explain the removal of such introns, recognition of the exons instead of recognition of the introns has been proposed. This thesis studies this exon definition mechanism using a bottom-up approach. To reduce the complexity of the system under study, this exon definition mechanism was addressed using designer exons made up of prototype sequence modules of our own design (including an exonic splicing enhancer or silencer). Studies were performed in vitro with a set of DEs obtained from random combinations of the exonic splicing enhancer and the exonic splicing silencer modules. The results showed considerable variability both in terms of the composition and size of the DEs and in terms of their inclusion level. To understand how different DEs generated different inclusion levels, the problem was divided into understanding separately parameters varied between DEs. Subsequent studies focused on each of three parameters: size, ESE composition and ESS composition. The final objective was to be able to combine their effects to predict the inclusion levels obtained for the "random" DEs mentioned previously. To complement this experimental approach an equation was generated in two stages. First a general "framework" equation was obtained modeling a necessary exon definition complex that enabled commitment to inclusion. This equation used rates for the formation and dissociation of this complex without elaborating on the details for how those rates came about. In the second stage, however, formation and dissociation were modeled using novel but intuitive ideas and these models were combined into a final equation. This equation using the single-parameter perturbation data obtained previously performed well in predicting the inclusion levels of the "random" DEs. Additionally, both the final equation and the mechanisms proposed align well with results published by other groups. In order to make these results more accessible and to open more opportunities to extend them, an initial attempt is presented to identify the proteins involved in the functionality observed for each of the sequences used.

The ribosome is responsible for mRNA-templated protein translation in all living cells. The translational machinery (TM) has evolved to use 20 amino acids each esterified onto one of several tRNA bodies. While the active site of the ribosome, known as the peptidyl transferase center (PTC), is able to handle a remarkable amount of substrate diversity, many classes of unnatural amino acids are not compatible with the TM. For example, in the field of unnatural amino acid mutagenesis, the site-specific incorporation of biologically useful amino acids into proteins, such as fluorophores, has often proven to be unfeasible. This runs counter to the accepted notion that the ribosome is blind to the structure of the amino acid and is capable of accepting any amino acid as long as the mRNA codon: tRNA anticodon pairing is correct. Two studies by our group set out to test the hypothesis that the ribosome can indeed discriminate the structure of the amino acid. Using a fully purified E. coli translation system, the first study showed that natural amino acids misacylated onto fully modified but non-native tRNAs show small but reproducible effects on the steps of aminoacyl-tRNA (aa-tRNA) selection. The second study, in which I participated, utilized D-aa-tRNAs in the same E. coli translation system to study how amino acids of the inverted stereochemistry to those found in ribosomally-synthesized proteins affect translation elongation. We showed that these unnatural substrates serve as peptidyl acceptors but once translocated into the P-site of the ribosome, fail as peptidyl donors and stall translation elongation by inactivating the PTC. The motivation of my work has been to further characterize the effects of D-aa-tRNAs on translation elongation. To this end, I examined how the PTC is affected structurally and functionally by the presence of ribosomal substrates containing D-amino acids. Chapter one contains an introduction to this work. Chapter two describes chemical probing experiments that demonstrate that the presence of peptidyl-D-aminoacyl-tRNAs in the P-site of the ribosome allosterically modulates the secondary structure of ribosomal exit tunnel nucleotides A2058 and A2059. Chapter three describes how the reactivity of peptidyl-D-aminoacyl-tRNAs to form tripeptides is highly dependent on the identity of the amino acid it is reacting with; protein yields can be close to what is obtained with natural amino acids or almost completely abolished. Chapter four contains the methods used to do this research. From the observations presented here as well as from the work of other laboratories, a picture of the PTC emerges in which the pairing of the A- and P- site substrates is integral in either promoting or suppressing catalysis by the PTC. This work has implications for the field of unnatural amino acid mutagenesis, particularly for strategies to improve the incorporation of interesting unnatural amino acid by the ribosome. In addition, this work adds an important aspect to the growing body of knowledge of ribosome stalling at the PTC.

Ribosomes are universally conserved macromolecular machines found within all living cells that catalyze protein synthesis, one of nature’s most fundamental processes. Ribosomes synthesize proteins, which are polymeric chains of amino acids, by incorporating the amino acids one at a time via aminoacylated-transfer RNAs (aa-tRNAs), based on translation of the sequence of triplet- nucleotide codons presented by the messenger RNA (mRNA) template that is a direct readout of genomic DNA. Recent biochemical, structural, dynamic, and computational studies have uncovered large-scale conformational changes of the ribosome, its tRNA substrates, and the additional protein translation factors that play important roles in regulating protein synthesis, especially during the elongation phase of translation when the bulk of each protein is synthesized. How the ribosome, its translation elongation factors, tRNAs, and mRNA physically coordinate and regulate the movements of the tRNAs carrying amino acids into, through, and out of the ribosome remains one of the more fundamental questions in the mechanistic studies of protein synthesis. A complete understanding of the conformational dynamics of ribosomal complexes will improve our knowledge of how translation is regulated, including how ribosome-targeting antibiotics regulate translation elongation, and will provide crucial information for designing next-generation antibiotics. In this thesis I have investigated the conformational dynamics of the ribosome during the elongation phase of protein synthesis at the single-molecule level using single-molecule fluorescence resonance energy transfer (smFRET) microscopy experiments. Specifically, I have studied ribosomal dynamics during the elongation phase of translation in the presence of a tRNAPro in the context of an mRNA that has the propensity to shift out of the reading frame. My studies have revealed information about the mechanistic and regulatory functions of the posttranscriptional modifications of tRNAPro in a context in which the ribosomal complex has the propensity to undergo non-programmed +1-frameshifting, in which the tRNA-mRNA base pairing shifts one base toward the 3’ end of the mRNA, and if unchecked, leads to the synthesis of a polypeptide with a completely different sequence of amino acids. My data suggests that in this context, the mechanism underlying non-programmed +1-frameshifting involves the tRNA shifting out of frame prior to the tRNA being accommodated in the P site, i.e. either while the tRNA is in the A site, or more likely, during translocation of the tRNA from the A site to the P site, and not while the tRNA is already occupying the P site, as previously proposed.