Deficiency in both type I and type II DNA topoisomerase activities differentially affect rRNA and ribosomal protein synthesis in Schizosaccharomyces pombe
01 Apr 1988-Current Genetics (Springer-Verlag)-Vol. 13, Iss: 4, pp 305-314
TL;DR: Observations indicate the apparent lack of tight coupling between rRNA and r-protein synthesis in S. pombe under these experimental conditions.
Abstract: The synthesis of rRNA and r-proteins was studied in temperature-sensitive topoisomerase mutants of the fisson yeast Schizosaccharomyces pombe. To reduce the severity of heatshock response seen in the wild type strain, slow temperature shift-up of the cultures was used to inactivate the mutant topoisomerases. It was found that the temperature shift caused a large preferential reduction of rRNA synthesis in the top1top2 double mutant. In contrast, no preferential inhibition of rRNA synthesis was observed in top1 or top2 single mutants, although some reduction in the total RNA synthesis was observed in the top2 mutant. Thus, as observed with Saccharomyces cerevisiae (Brill et al. 1987), relaxation of supercoiled DNA structures by either topoisomerase I or II appears to be essential for efficient transcription of rRNA genes. Analysis of r-protein synthesis indicated that there were small decreases in the differential synthesis rates of r-proteins after temperature shift-up in the top1top2 mutant, but the observed negative effects on r-protein synthesis was much smaller than that on rRNA synthesis, and degradation of the newly synthesized r-proteins was observed. These observations indicate the apparent lack of tight coupling between rRNA and r-protein synthesis in S. pombe under these experimental conditions.
TL;DR: Unlike most types of cells, yeast cells appear to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosome, causing slow growth.
Abstract: The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32, the protein itself causes a feedback inhibition of the splicing of the transcript of its own gene. The synthesis of ribosomes involves a massive transfer of material across the nuclear envelope in both directions. Nuclear localization signals have been identified for at least three ribosomal proteins; they are similar but not identical to those identified for the simian virus 40 T antigen. There is no information about how ribosomal subunits are transported from the nucleus to the cytoplasm.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: This chapter discusses the DNA topoisomerases as anticancer drug targets and the signals and events involved in cell killing, in particular the early signals induced by drug-mediated DNA damage.
Abstract: Publisher Summary This chapter discusses the DNA topoisomerases as anticancer drug targets. Much progress has been made in recent years in understanding the mechanism of action of antitumor drugs that target topoisomerases. However, while it is now well established that these drugs interact with the cleavable complex, molecular details of the protein-DNA-drug interaction are only just starting to emerge. The large body of information available from the study of drug analogs has provided some detailed information on the structural requirements for drugs to interact successfully with the cleavable complex. Little is known about the actual cell killing mechanism. This question, which involves events beyond cleavable complex formation and its interaction with drugs, is becoming more and more important. Knowledge of the signals and events involved in cell killing, in particular the early signals induced by drug-mediated DNA damage, might eventually lead to the discovery and identification of new targets for antitumor drugs.
TL;DR: The DNA topoisomerase-dependent excision/integration of r DNA is discussed in terms of the possibility of rDNA supercoiling by transcription and the effects of DNA topology on intra- and interchromosomal recombination.
Abstract: In a yeast DNA topoisomerase double mutant TG205 (Δ top1 top2–4 ), over half of the rDNA is present as extrachromosomal rings containing one 9 kb unit of the rDNA gene or tandem repeats of it. Expression of a plasmid-borne TOP1 or TOP2 gene in the strain leads to the integation of the extrachromosomal rDNA rings back into the chromosomal rDNA cluster. When the plasmid-borne topoisomerase gene is expressed from an inducible promoter of the GAL1 gene, repression of the gene by dextrose leads to reappearance of the extrachromosomal rDNA rings. The DNA topoisomerase-dependent excision/integration of rDNA is discussed in terms of the possibility of rDNA supercoiling by transcription and the effects of DNA topology on intra- and interchromosomal recombination.
TL;DR: In celebrating the centennial of the American Society for Microbiology, many people will surely recall the central importance that research using microbial systems played in the birth and the subsequent development of molecular biology in the latter half of the 100-year history.
Abstract: In celebrating the centennial of the American Society for Microbiology, many people will surely recall the central importance that research using microbial systems played in the birth and the subsequent development of molecular biology in the latter half of the 100-year history. Starting from the demonstration of DNA as the genetic material, a series of key experiments, such as the proof of semiconservative replication of DNA, the discovery of mRNA as the information carrier between DNA and protein, and the eventual elucidation of the genetic code, were done mostly with microbial systems, the enteric bacterium Escherichia coli and its bacteriophages in particular. These basic principles in molecular genetics discovered with bacterial systems soon proved to be true for almost all organisms. Consequently, early research activities in molecular biology were concentrated on E. coli and related bacterial and phage systems, generating the initial attitude of many molecular biologists reflected in the well-publicized phrase, “What is true for E. coli is true for elephants.” (The acceptance of such an attitude at that time was not very surprising. Prior to the successful development of molecular biology, research in the field of intermediary metabolism from the 1920s through 1940s had demonstrated abundant evidence for the unity of biochemistry from microorganisms to humans, e.g., the mechanism of energy [ATP] production and its use for anabolic reactions [see also reference 42;]. Starting my first research as a student of fermentation biochemistry in 1950, I was certainly influenced by the prevalent belief, the unity of biochemistry, at that time.) Of course, in view of the bewildering diversity known in biology, especially some fundamental differences between prokaryotes and eukaryotes or single-cell versus multicellular organisms, such a view was expected to be too simple and naive. Thus, it was soon realized that the actual mechanisms and principles underlying certain biological functions, including diverse modes found in regulation of gene expression, are the consequences of evolutionary tinkering and may not necessarily be universal among diverse organisms (for a detailed discussion on evolution and tinkering, see reference 27). Nevertheless, attempts to extend factual observations or concepts obtained in one system (e.g., prokaryotes) to another (e.g., eukaryotes) have been made repeatedly and often turned out to be stimulating if not successful. As a person who was engaged in studies of synthesis of ribosomes and ribosomal components first in E. coli and later in Saccharomyces cerevisiae, I will recount some of the research activities on this subject which I have touched upon in this context.
TL;DR: Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribOSomal sub unit protein, rp59, to determine the effects of depriving cells of either of these Ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomic proteins and ribosom RNAs.
Abstract: Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.
TL;DR: A comparison of different Organisms and Stages of Development and Heat-Induced Lethality and Thermotolerance and the role of RNA Processing are presented.
Abstract: PERSPECTIVES AND SUMMARY . . . . . 1151 CHARACTERIZATION OF THE RESPONSE 1153 Comparison: Different Organisms and Stages of Development. ll53 The Proteins Induced by Heat ... . 1155 RNAs Induced by Heat 1167 OTHER INDUCTIONS OF HSPs 1168 Developmental Inductions .. . ... . . . . . . .. .. .. . ... ...... . .... . .. . . . 1168 Other Inducers 1 1 69 Is There a Common Mechanism? ........ ... 1170 GENOME ORGANIZATION 1172 REGULATION OF THE RESPONSE 1173 Transcription 1173 Translation . . . . .. ..... ......... .. . . .. .. .. .... . .. ...... . . . .. . . . . .. .. .. . .. .. .. . . . . . . . . . 1177 RNA Processing 1178 TOLERANCE TO HEAT AND OTHER FORMS OF STRESS . . . 1179 Heat-Induced Lethality and Thermotolerance . . . . . . . . . . . . . 1179 Phenocopies 1182 CONCLUDING REMARKS 1184
TL;DR: Physiological characterisation of the mutants has shown that DNA synthesis and nuclear division form a cycle of mutually dependent events which can operate in the absence of cell plate formation.
Abstract: Twenty seven recessive temperature sensitive mutants have been isolated in Schizosaccharomyces pombe which are unable to complete the cell division cycle at the restrictive temperature. These mutants define 14 unlinked genes which are involved in DNA synthesis, nuclear division and cell plate formation. The products from most of these genes complete their function just before the cell cycle event in which they are involved. Physiological characterisation of the mutants has shown that DNA synthesis and nuclear division form a cycle of mutually dependent events which can operate in the absence of cell plate formation. Cell plate formation itself is usually dependent upon the completion of nuclear division.
TL;DR: This chapter discusses the regulation of Ribosomal Protein Genes and their Transcription in the context of ribosomal RNA regulation.
Abstract: CONTENTS PERSPECTIVES AND SUMMARY 75 REGULATION OF RIBOSOMAL PROTEIN SyNTHESIS 77 Organization of Ribosomal Protein Genes ...... 78 Translational Feedback Regulation 78 Transcription of Ribosomal Protein Genes and I ts Regulation 91 REGULATION OF rRNA AND RIBOSOME SyNTHESIS 9S Organization ofrRNA Genes and Their Transcription 96' Stringent Control 99 Growth Rate-Dependent Control 101 O ther Possible Modes of R egulation 109
01 Jan 1986
TL;DR: The papers in this book cover the Ribosome conference held at the University of Texas Marine Science Institute and topics covered include: Structure of ribosomes; Self-organization ofribosomal RNA; Structural dynamics of the translating ribosome; and Mechanism of Ribosomes Translocation.
Abstract: The papers in this book cover the Ribosome conference held at the University of Texas Marine Science Institute. The topics covered include: Structure of ribosomes; Self-organization of ribosomal RNA; Structural dynamics of the translating ribosome; and Mechanism of Ribosome Translocation.
TL;DR: The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.
Abstract: We have isolated mutants defective in DNA topoisomerases and an endonuclease from the fission yeast Schizosaccharomyces pombe by screening individual extracts of mutagenized cells. Two type I topoisomerase mutants (top1) and three endonuclease mutants (end1) were all viable. The double mutant top1 end1 was also viable and, in its extract, Mg2+- and ATP- dependent type II activity could be detected. Three temperature-sensitive (ts-) mutants having heat-sensitive (hs-) type II enzymes were isolated, and the ts- marker cosegregated with the hs- type II activity. All the ts- mutations fell in one gene (top2) tightly linked to leul in chromosome II. The nuclear division of single top2 mutants was blocked at the restrictive temperature, but the formation of a septum was not inhibited so that the nucleus was cut across with the cell plate. In contrast, the double top1 top2 mutants were rapidly arrested at various stages of the cell cycle, showing a strikingly altered nuclear chromatin region. The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.