No description available.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

RNA has many diverse functions in living organisms, from serving as genome for many viruses, to regulating DNA replication, transcription, translation and other metabolic processes. Some RNA, like protein, has been shown to have catalytic activity. The great proportion of the mass of RNA in living cells, in the form of ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), constitutes the machinery of protein synthesis, the remainder (approximately 2%) consists of many heterogeneous RNA species of relatively small size, loosely termed "small RNAs", the functions of many of which are completely unknown. In an attempt to understand some of these functions, three hitherto undescribed small RNAs of the budding yeast Saccharomyces cerevisiae were identified and their genes were cloned. These three small RNAs, which lack polyadenylation at their 3' ends, appear to represent the three most abundant RNA species in this organism after rRNA and tRNA. The most abundant of the three was found to be mainly cytoplasmic and was therefore called "small cytoplasmic RNA 1" (scR1). The other two RNAs, named snR17 and snR30, were found to be enriched in nuclear fractions and to possess trimethyl guanosine cap structures at their 5 ends, identifying them as belonging to the ubiquitous class of "U" small nuclear RNAs (U snRNAs), of which several are required for the endonucleolytic cleavage and splicing reactions in the maturation pathways of nuclear precursor mRNAs (pre-mRNA). Whereas scR1 and snR30 are both encoded by single genes, snR17 is the only yeast small RNA found so far to be encoded by two genes. SnR17 was found to be essential: haploid yeast strains lacking intact copies of one or other of the genes appeared to grow normally, but strains lacking both genes were inviable. The nucleotide sequences of the snR17 genes were determined, and the primary and predicted secondary structures of the RNA, 328 nucleotides in length, were found to show significant similarities to those of U3 snRNA, an abundant U snRNA, the function of which is not known. SnR17 belongs to a family of S. cerevisiae snRNAs which, unlike those involved in pre-mRNA splicing, are located in the nucleolus hydrogen-bonded to pre-rRNA, and are associated with antigenic protein that is recognized by human antibodies specific for a 36 kD polypeptide of the U3 small nuclear ribonucleoprotein (U3 snRNP) in mammals. U3 snRNA is also nucleolar and associated with pre-rRNA. Given their structural similarities, snR17 and U3 snRNA are presumably homologous. Yeast snRNAs associated with the anti-(U3)RNP antigen share with U3 snRNAs a conserved nucleotide sequence element. This sequence element alone, however, when injected into Xenopus oocytes, was not sufficient to direct binding of the antigen. The association of snRNAs with pre-rRNA suggests that they function in ribosomal biogenesis.

BE [(1,4)-a-D-glucan:(I,4)-a-D-glucan 6-glucosyltransferase, EC 2.4.1.18] catalyses a transglycosylation reaction where a branch-point is created by the cleavage of an a-l,4 glycosidic bond to form an a-l,6 glycosidic bond. Branching enzyme (BE) from baker's yeast was purified to near homogeneity by chromatography on DEAE-cellulose, Sephacryl S-200 and Protein Pak Q. Electrophoresis on SDS-PAGE revealed one major band of molecular weight 74 kDa. Three distinct methods for determining BE activity (Phosphorylase Stimulation, Iodine- Binding and Branch-Linkage Assays) were used to characterise the purified protein. The enzyme displayed a temperature optimum between 15-25°C and a broad pH optimum of 6.5-7.5 with maximum activity occurring in phosphate buffer. The enzyme was fully stable after incubation at 20°C for 5 hours. A Km value of 1474 Jlg/ ml for amylose was obtained. Primary structural analysis involving N-terminal sequencing and amino acid composition suggested that yeast BE may share some homology with BEs isolated from other sources. Immunological comparisons between yeast, maize (BEll) and Escherichia coli BE using yeast polyclonal antiserum indicated that the enzymes may share antigenic determinants. However, similar comparisons between yeast BE and E.coli antiserum revealed that the antibody only recognised yeast BE in its denatured conformation. Yeast BE was used to modify potato amylose and amylopectin and wheat starch. The enzyme was capable of introducing additional branch points to these substrates resulting in a displacement of the iodine Amax from 629 nm to 568 nm, from 543 nm to 411 nm and from 632 nm to 568 nm for amylose, amylopectin and wheat starch, respectively. HPAEC-PAD analysis of the branched products produced by yeast BE revealed that predominantly short chains of dp 2 to I? were transferred. At least three BE fractions of higher specific activities were isolated from brewer's yeast hatyested at the late exponential phase, suggesting the expression of more than one BE in Saccharomyces cerevisiae.

No description available.

No description available.

Eukaryotic ribosome synthesis is a complex process that consumes a lot of energy and involves several hundreds of trans-acting factors that transiently associate with nascent ribosomes. Biogenesis of ribosomal subunits (the small 40S and the large 60S) starts with transcription of a long precursor ribosomal RNA (pre-rRNA) by RNA polymerase I (Pol I) in the nucleolus. This is a key step that globally controls yeast ribosome synthesis. The pre-rRNA, ‘the 35S transcript’, encodes the mature sequence (18S, 5.8S, and 25S) rRNA constituents of both the 40S and 60S subunits. The 35S transcript is subsequently modified, cleaved (processed) and assembled with numerous structural ribosomal proteins and ribosome synthesis factors (trans-acting factors) to form various ribosomal particles (pre-ribosomes, precursors to the 40S and 60S subunits) along ribosome assembly pathway. In the budding yeast Saccharomyces cerevisiae, it has been reported recently that the processing of the 35S nascent transcript and the assembly of pre-ribosomes occur concomitantly with Pol I transcription in the nucleolus. In this process, the growing Pol I transcript gradually assembles with pre-40S structural ribosomal proteins and ribosomal synthesis factors to form the so-called ‘SSU-processome’ or ‘90S pre-ribosome’, the earliest precursor of the 40S subunit. The SSU-processome/90S pre-ribosome localizes to the nucleolus and consists of the 35S pre-rRNA, the U3 small nucleolar (sno) RNA, about a dozen of 40S ribosomal proteins and more than forty ribosome synthesis factors. The U3 snoRNA and pre-40S ribosome synthesis factors are all implicated in the processing of the 35S precursor (at sites A0, A1 and A2) and therefore in the synthesis of the 18S rRNA component of the 40S subunit. Significantly, the association of the U3 snoRNA with the growing 35S transcript is important for pre-40S assembly, whereas its dissociation from the processed transcript (following cleavage at sites A0-A2) is crucial for the overall structural remodeling of the 18S rRNA and for the formation of pre-40S ribosomes from the earliest precursor 90S particles. This thesis mostly addresses the identification and functional characterization of Esf2 and Bfr2, two novel 40S synthesis factors, components of the SSU-processome/90S pre-ribosome in yeast. Both proteins localize to the nucleolus and their genetic depletions lead to failure in the production of 40S subunits. In the absence of either factor, the 35S pre-rRNA is not processed at sites A0-A2 and the 18S rRNA is not synthesized. Also, pre-ribosome assembly is affected and pre-40S ribosomes fail to mature properly. Strikingly, in the absence of either factor, the U3 snoRNA remains associated with unprocessed 35S transcript within pre-ribosomes indicating that Esf2 and Bfr2 are required to dissociate U3 from pre-ribosomes. This process also involves RNP (ribonucleoprotein particle) unwinding activities of the putative RNA helicase Dbp8. La biogenèse du ribosome eucaryote est un processus complexe qui consomme beaucoup d’énergie et implique plusieurs centaines de facteurs trans qui s’associent de manière transitoire avec les pré-ribosomes en cours de formation. La biogenèse des sous-unités ribosomiques (la petite sous-unité 40S et la grande sous-unité 60S) débute dans le nucléole par la synthèse d’un long précurseur d’ARN ribosomique (le pré-ARNr, dit 35S chez la levure Saccharomyces cerevisiae) par l’ARN Polymérase I (Pol I). Ceci constitue une étape clé dans le contrôle global de la synthèse du ribosome chez la levure. Le pré-ARNr 35S renferme les séquences des ARNr matures 18S (ARNr de la sous-unité 40S) et 5.8S et 25S (deux des trois ARNr de la sous-unité 60S). Le pré-ARNr 35S subit un long processus de maturation et d’assemblage au cours duquel il est modifié, clivé (on parle du « processing » du pré-ARNr) et s’assemble avec des protéines ribosomiques (« RP », composants structuraux des sous-unités ribosomiques matures) et de nombreux facteurs de synthèse (facteurs trans) pour former différentes particules pré-ribosomiques (précurseurs des sous-unités 40S et 60S). Chez la levure S. cerevisiae, il a récemment été montré que le processing du pré-ARNr 35S et l’assemblage des pré-ribosomes se produisent de manière concomminante avec la transcription Pol I dans le nucléole. Ainsi, le transcrit Pol I en cours de synthèse s’assemble progressivement avec des facteurs de synthèse ainsi que des RP pour former le « SSU processome » ou « pré-ribosome 90S », tout premier précurseur de la petite sous-unité 40S. Le SSU processome/pré-ribosome 90S est localisé dans le nucléole et est consisté du pré-ARNr 35S naissant, du petit ARN nucléolaire (snoRNA) U3, d’une douzaine de RP de la petite sous-unité 40S et de plus de 40 facteurs de synthèse. Le snoRNA U3 et ces facteurs de synthèse sont tous impliqués dans les clivages du pré-ARNr 35S aux sites A0, A1 et A2, et donc dans la biogenèse de l’ARNr 18S. L’association du snoRNA U3 avec le pré-ARNr 35S naissant est importante pour l’assemblage du SSU processome/pré-ribosome 90S. Par ailleurs, sa dissociation après les clivages aux sites A0-A2 permet un remodelage structural général de l’ARNr 18S et la formation du « pré-ribosome 40S » à partir de la particule précoce 90S. Au cours de cette thèse, nous avons identifié et caractérisé fonctionnelement chez la levure deux nouveaux facteurs de synthèse de la petite sous-unité 40S et composants du SSU processome/pré-ribosome 90S: Esf2 et Bfr2. Ces deux protéines sont localisées dans le nucléole. Leur déplétion entraîne une incapacité à produire la sous-unité ribosomique 40S. En l’absence d’Esf2 ou Bfr2, le pré-ARNr 35S n’est plus clivé aux sites A0-A2 et l’ARNr 18S mature n’est plus produit. L’assemblage des pré-ribosomes est aussi affecté, notamment la formation du pré-ribosome 40S. De manière importante, en l’absence de l’un ou l’autre de ces facteurs, le snoRNA U3 reste associé au pré-ARNr 35S non clivé au sein des pré-ribosomes, indiquant qu’Esf2 et Bfr2 sont requises pour la dissociation d’U3 des pré-ribosomes. Ce processus implique aussi Dbp8, une hélicase à ARN présumée.

No description available.

No description available.

No description available.