5. Yeast Genetic Structures and Functions
- Prof. Dr. Horst Feldmann1,2
Published Online: 26 SEP 2012
Copyright © 2012 Wiley-VCH Verlag GmbH & Co. KGaA
Yeast: Molecular and Cell Biology, Second Edition
How to Cite
Feldmann, H. (ed) (2012) Yeast Genetic Structures and Functions, in Yeast: Molecular and Cell Biology, Second Edition, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527659180.ch5
Adolf Butenandt Institute, Molecular Biology, Ludwig-Maximilians-Universität M¨nchen, Schillerstr. 44, 80336 M¨nchen, Germany
Ludwig-Thoma-Strasse 22B, 85232 Bergkirchen, Germany
- Published Online: 26 SEP 2012
- Published Print: 22 AUG 2012
Print ISBN: 9783527332526
Online ISBN: 9783527659180
- yeastgenetic structures;
- chromosome structure;
- yeast mitochondrial genome
• This chapter is devoted to a thorough description of the genetic entities and their functions that have been generated by nature to make a small unicellular organism like yeast work as independently and successfully as possible. Some of these structures were recognized early in yeast molecular biology; refinements regarding their functions and interplay became visible only recently.
• At a molecular level, functional sites along yeast chromosomes as well as extrachromosomal elements were characterized. It was observed that the centromeric DNA sequences in all yeast chromosomes – the sites of kinetochore formation and chromosome attachment to mitotic and meiotic spindles – extend over only 200 bp, contrary to the much larger, complex centromeres in S. pombe or mammalian cells.
• As early as in 1979, it was detected that particular short chromosomal fragments would lend circular plasmids the power to autonomously replicate. Similar copies of these elements were found to occur frequently along all yeast chromosomes. More intense studies then led to the identification of short loci (ARSs) that form binding sites for the ORC protein complex. Such “prereplicative” complexes are assembled during the M and G1 phases, persist during the cell cycle, and serve as “markers” for replication origins during the S phase. “Firing” of an origin is initiated by recruitment of the ATP-dependent protein Cdc6 to the ORC complex, which in turn loads other necessary components for chromosome duplication on to chromatin. We consider events that lead to DNA damage and how such obstacles are eliminated. Here, we concentrate primarily on DNA replication and discuss the cell cycle in Chapter 7.
• As a prerequisite for understanding chromosome structure at the most detailed molecular level, the organization of chromatin structure including the histones is discussed. The past years of research have resulted in the discovery of numerous modifications on histones and the enzymes responsible for their deposition. In turn, modifications of histones invoke a reorganization of chromatin structure effected by numerous remodeling complexes, resulting in the promotion or silencing of gene activity.
• In the 1930s, pioneering work by Muller with flies and by McClintock with maize led to the description of telomeres as structures that protect chromosomes from loss and end-to-end fusions. The first telomeres from yeast were cloned in linear plasmid vectors in 1982 and have since served as an indispensable model. Generally, the ultimate ends of eukaryotic chromosomes are composed of reiterated short (G-rich) sequences that bear similarity among different organisms. Owing to their “open-end” structure, a special set of factors is required for their noncanonical, RNA-templated replication. Telomeric DNA and its affiliated proteins serve two crucial functions – they lend stability to the single chromosomes and their structure prevents telomeres from being confused with damaged DNA by checkpoint activities, whose downstream effectors could promote their fusion or degradation, eventually leading to cell cycle arrest and/or cell death. This cell cycle-regulated degradation has been best demonstrated in S. cerevisiae, but it probably also occurs in higher eukaryotes.
• The occurrence of transposable elements in yeast was established in 1979. Since then, five different types of retrotransposons have been identified, all of which bear high similarity to retroviruses; some of them were shown to be propagated via VLPs. In contrast to retroviruses, however, these entities are not infectious. Rather, they attracted much attention because they are associated with DNA rearrangements and could be used as models for host–parasite interactions.
• In further sections, we focus on the structures and properties of the yeast cellular RNAs: tRNAs, rRNAs, and mRNAs. In the mid-1960s, efforts to elucidate the genetic code raised an interest in determining the primary structures of tRNAs – those molecules that had been postulated by Francis Crick to function as adaptors in protein synthesis. In 1960, Monier, Stephenson, and Zamecnik devised an approach to isolate low-molecular-weight RNA from yeast by simple phenol extraction and precipitation of the soluble RNA from the aqueous phase with ethanol, which made this organism a most useful source for further work. Fractionation and subsequent analysis of purified tRNA species was much more tedious and took several years. In the end, the first sequence of a tRNA to be determined in 1965 was that of an alanine-specific tRNA from yeast, followed by the sequences of yeast serine, tyrosine, and phenylalanine tRNAs. In all cases, these sequences could be arranged in a “clover-leaf” structure, with the anticodon triplet exposed in the anticodon loop. Some 10 years later, the three-dimensional structure of yeast phenylalanine tRNA was resolved. This model formed a basis to investigate the interactions of tRNA with its cognate partners – the amino acid tRNA synthetases and nucleotide-modifying enzymes. The newly developed molecular techniques, such as cloning and sequencing DNA, were successfully applied to study the genomic arrangement of yeast tRNA genes and to follow the biogenesis of mature tRNA from their precursors.
• Research in 1977 was highlighted by the detection of introns in mammalian mRNAs, but it came as a similar surprise in the same year that yeast tRNA genes also contain “intervening sequences” that have to be processed out from the transcripts during maturation (a procedure that later was confirmed for many eukaryotic tRNA genes in general). Although only some 20% of the nuclear yeast tRNA genes were later recognized to possess intervening sequences, tRNA precursors could successfully be used to characterize the enzymes involved in the cleavage and ligation reactions.
• Although research on yeast ribosomes and ribosome synthesis started in the early 1970s, the fundamental knowledge to this field was mainly contributed from the studies of prokaryotes, preferably the bacterium E. coli, or mammalian cells. Nonetheless, the yeast system provided useful details on eukaryotic ribosomal components, on their maturation, and on the regulation of ribosome biosynthesis.
• A field to which yeast made significant contributions was to unravel the mechanism of splicing of eukaryotic pre-mRNAs. Although only 4–5% of the protein-encoding genes from yeast possess introns, a comparison of the “splice cycle” in yeast and mammals revealed great similarity. This finding again underlined the notion that basic cellular mechanisms and components have been conserved throughout evolution. It took about 15 years (1984–1998) to work out a detailed picture, but there are still novel features to be detected. The spliceosome was recognized as a particle in which the RNA components (pre-mRNA as the substrate and auxiliary snRNAs) were associated with particular proteins (PRPs) forming stable subcellular RNA–protein complexes during the splicing process. In all, over 100 such proteins were characterized. Of invaluable help in defining the single steps within the cycle were a multitude of PRP mutants from yeast, which were defective in splicing because of their inability to assemble specific subcomplexes.
• In addition to the killer plasmids and the 2 µm plasmid, S. cerevisiae harbors several unusual, protein-based genetic elements that have been classified as prions. The first two were detected in 1965 and 1971, but ongoing experiments point to the existence of further such elements in yeast.
• Last, but not least, a final section is devoted to the mitochondrial genome, whose organization brought about some surprises. Yeast mitochondria can be obtained as respiratory-competent entities, permitting a functional dissection of respiration, oxidative phosphorylation, and protein import; details of mitochondrial function are presented in Chapter 11.