No abstract

The viroid of the potato spindle tuber disease (PSTV) is a covalently closed ring of 359 ribonucleotides. As a result of intramolecular base pairing, a serial arrangement of double-helical sections and internal loops form a unique rod-like secondary structure. PSTV is the first pathogen of a eukaryotic organism for which the complete molecular structure has been established.

No abstract

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

In a genetic screen aimed at the identification of trans-acting factors involved in mRNA 3-end processing of budding yeast, we have previously isolated two temperature-sensitive mutants with an apparent defect in the 3-end formation of a plasmid-derived pre-mRNA. Surprisingly, both mutants were rescued by the essential gene ESS1/PTF1 that encoded a putative peptidylprolyl-cis/trans-isomerase (PPIase) (Hani, J., Stumpf, G., and Domdey, H. (1995) FEBS Lett. 365, 198 -202). Such enzymes, which catalyze the cis/trans-interconversion of peptide bonds N-terminal of prolines, are suggested to play a role in protein folding or trafficking. Here we report that Ptf1p shows PPIase activity in vitro, displaying an unusual substrate specificity for peptides with phosphorylated serine and threonine residues preceding proline. Both mutations were found to result in amino acid substitutions of highly conserved residues within the PPIase domain, causing a marked decrease in PPIase activity of the mutant enzymes. Our results are suggestive of a so far unknown involvement of a PPIase in mRNA 3-end formation in Saccharomyces cerevisiae.Despite intensive efforts to unravel the complex process of mRNA 3Ј-end formation in Saccharomyces cerevisiae, the list of participating factors still awaits its completion.We have recently isolated a gene complementing the phenotype of two temperature-sensitive yeast mutants that were impaired in mRNA 3Ј-end formation. This gene, designated PTF1 (processing/termination factor 1; identical with the previously described ESS1 (1)), encodes a protein that, by virtue of sequence similarity, was identified as a peptidylprolyl-cis/ trans-isomerase (PPIase) 1 (2). PPIases are ubiquitous enzymes that catalyze the interconversion from cis to trans of peptide bonds preceding a proline and are thought to accelerate this often rate-limiting step in the folding of a number of proteins in vivo (3-6).PPIases are divided in three families, based on their sensitivities toward two clinically relevant immunosuppressants: the cyclosporin A-binding proteins (cyclophilins), the FK506-binding proteins, and a third family, named after the Escherichia coli protein parvulin, which is not inhibited by either of the two drugs (for review see Refs. 3-6). In addition, the members of each family are characterized by conserved but distinct amino acid domains. By this criterion, PTF1 was predicted to belong to the parvulin family of PPIases (2).Although disruption of PPIase genes did not generally impair cell growth (7-8), PTF1 was the first PPIase gene shown to be essential for cell viability (1). In fact, PTF1 is the only essential PPIase gene in S. cerevisiae as demonstrated more recently by the viability of a yeast mutant lacking the remaining 12 PPIases, members of the other two immunosuppressant binding families. (8). So far, the only other example of an essential PPIase is the recently discovered PIN1, a human protein, that is structurally and functionally related to Ptf1p (9 -10).In this paper we describe the genetic screen ...

The complete nucleotide sequence of citrus exocortis viroid (CEV, propagated in Gymura) and chrysanthemum stunt viroid (CSV, propagated in Cineraria) has been established, using labelling in vilro and direct RNA sequencing methods and a new screening procedure for the rapid selection of suitable RNA fragments from limited digests.The covalently closed circular single-stranded viroid RNAs consist of 371 (CEV) and 354 (CSV) nucleotides, respectively. As previously shown for potato spindle tuber viroid (PSTV, 359 nucleotides), CEV and CSV also contain a long polypurine sequence. Maximal base-pairing of the established CEV and CSV sequences results in an extended rod-like secondary structure similar to that previously established for PSTV and as predicted from detailed physicochemical studies of all these viroids. Although the three viroid species sequenced to date differ in size and nucleotide sequence, there is 60-73 homology between them. As PSTV, CEV and CSV also contain conserved complementary sequences which are separated from each other in the native secondary structure. We postulate that the resulting 'secondary' hairpins, being formed and observed in vitro during the complex process of thermal denaturation of viroid RNA, must have a vital, although yet unknown, function in vivo. The possible origin and function of viroids are discussed on the basis of the characteristic structural features and of a considerable homology with U l a RNA found for a region highly conserved in the three viroids.

Extracts of wheat germ contain an RNA ligase activity that catalyses the conversation of linear polyribonucleotides into covalently closed circles. The newly formed 3',5'-phosphodiester bridge is resistant to alkali and a number of ribonucleases. This unusual stability is due to the presence of a phosphoryl group esterified to the 2'-position of the 3'-nucleotide joined. This is the first demonstration of an RNA ligase in higher plants and of a natural 2'-phosphomonoester, 3',5'-phosphodiester linkage.

No abstract