Structure analysis of the 5' external transcribed spacer of the precursor ribosomal RNA from Saccharomyces cerevisiae.

Full-length precursor ribosomal RNA molecules were produced in vitro using as a template, a plasmid containing the yeast 35 S pre-rRNA gene under the control of the phage T3 promoter. The higher-order structure of the 5'-external transcribed spacer (5' ETS) sequence in the 35S pre-rRNA molecule was studied using dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, RNase T1 and RNase V1 as structure-sensitive probes. Modified residues were detected by primer extension. Data produced were used to evaluate several theoretical structure models predicted by minimum free-energy calculations. A model for the entire 5'ETS region is proposed that accommodates 82% of the residues experimentally shown to be in either base-paired or single-stranded structure in the correct configuration. The model contains a high degree of secondary structure with ten stable hairpins of varying lengths and stabilities. The hairpins are composed of the Watson-Crick A.T and G.C pairs plus the non-canonical G.U pairs. Based on a comparative analysis of the 5' ETS sequence from Saccharomyces cerevisiae and Schizosaccharomyces pombe, most of the base-paired regions in the proposed model appear to be phylogenetically supported. The two sites previously shown to be crosslinked to U3 snRNA as well as the previously proposed recognition site for processing and one of the early processing site (based on sequence homology to the vertebrate ETS cleavage site) are located in single-stranded regions in the model. The present folding model for the 5' ETS in the 35 S pre-rRNA molecule should be useful in the investigations of the structure, function and processing of pre-rRNA.     

Structural analysis of the internal transcribed spacer 2 of the precursor ribosomal RNA from Saccharomyces cerevisiae

Full-length precursor ribosomal RNA molecules (6440 bases) were produced in vitro using a plasmid containing the yeast 35 S pre-rRNA operon under the control of phage T7 promoter. The higher-order structure of the internal transcribed spacer 2 (ITS-2) region (between the 5.8 S and 25 S rRNA sequence) in the pre-rRNA molecule was investigated using a combination of enzymatic and chemical structural probes. The data were used to evaluate several structural models predicted by a minimum free-energy calculation. The results supported a model in which the 3' end of the 5.8 S rRNA and the 5' end of the 25 S rRNA are hydrogen-bonded better than the one in which the ends are not. The model contains a high degree of secondary structure with several stable hairpins. Similar structural models for the ITS-2 regions of Schizosaccharomyces pombe, Saccharomyces carlsbergensis, mung bean and Xenopus laevis were derived. Certain common folding features appear to be conserved, in spite of extensive sequence divergence. The yeast model should be useful as a prototype in future investigations of the structure, function and processing of pre-rRNA.     

Some characteristics of processing sites in ribosomal precursor RNA of yeast.

The DNA sequences of the intergenic region between the 17S and 5.8S rRNA genes of the ribosomal RNA operon in yeast has been determined. In this region the 37S ribosomal precursor RNA is specifically cleaved at a number of sites in the course of the maturation process. The exact position of these processing sites has been established by sequence analysis of the terminal fragments of the respective RNA species. There appears to be no significant complementarity between the sequences surrounding the two termini of the 18S secondary precursor RNA nor between those surrounding the two termini of 17S mature rRNA. This finding implies that the processing of yeast 37S ribosomal precursor RNA is not directed by a double-strand specific ribonuclease previously shown to be involved in the processing of E. coli ribosomal precursor RNA [see Refs 1,2]. The processing sites of yeast ribosomal precursor RNA described in the present paper are all flanked at one side by a very [A+T]-rich sequence. In addition, sequence repeats are found around the processing sites in this precursor RNA. Finally, sequence homologies are present at the 3'-termini [6 nucleotides] and the 5'-termini [13 nucleotides] of a number of mature rRNA products and intermediate ribosomal RNA precursors. These structural features are discussed in terms of possible recognition sites for the processing enzymes.     

Specific processing of the bacterial beta-lactamase precursor in Saccharomyces cerevisiae

Synthesis and processing of the bacterial enzyme beta-lactamase (E.C. 3.5. 2.6) were studied in Saccharomyces cerevisiae. The 2-micron DNA vector pADH040-2 containing the yeast ADH1 promoter fused to the bacterial gene was used in order to obtain enhanced synthesis of the bacterial protein in yeast transformants. Both precursor and mature beta-lactamase were shown to be present in yeast cells, the precursor being the major product. The mature enzyme was purified about 500-fold over crude extracts to apparent homogeneity and thus represents nearly 0.2% of the total yeast protein. No difference in specific activity and molecular weight could be observed when compared with the authentic beta-lactamase from Escherichia coli. Specificity of the processing of beta-lactamase in yeast cells was verified by partial amino acid sequence analysis demonstrating the removal of the signal peptide at the correct position.     

Maturation of ribosomal precursor RNA in Saccharomyces cerevisiae: A mutant with a defect in both the transport and terminal processing of the 20 S species

A mutant (CLP-8) of Saccharomyces cerevisiae possesses an abnormal ratio of native ribosomal subunits since it has an apparent deficiency of cytoplasmic 40 S subparticles. The mutant also has an abnormal anti-association factor activity. The lesion(s) responsible for the ribosomal subunit inbalance is not temperature-sensitive and is incomplete since the mutant still grows, albeit at a reduced rate compared to that of its parent. The lesion(s) in CLP-8 is, however, expressed at the level of 20 S ribosomal precursor RNA maturation. Thus, relative to the wild-type strain, there is both a slowed transport of 20 S ribosomal precursor RNA from the nucleus to the cytoplasm and a slowed cytoplasmic conversion of this RNA component into the mature 18 S form.     

The HIV Tat protein affects processing of ribosomal RNA precursor

Background  

Inside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown.     

Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.