
Full text loading...
Category: Microbial Genetics and Molecular Biology
Ty1 and Ty5 of Saccharomyces cerevisiae, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817954/9781555812096_Chap26-1.gif /docserver/preview/fulltext/10.1128/9781555817954/9781555812096_Chap26-2.gifAbstract:
Ty1 and Ty5 of Saccharomyces cerevisiae are long terminal repeat (LTR) retrotransposons, members of a large and ubiquitous class of mobile genetic elements. Like the retroviruses, LTR retrotransposons replicate by reverse transcribing RNA into DNA and then integrating the DNA transposition intermediate into the genome of their host. This chapter begins with a description of Ty1 and Ty5, including their origins and genetic organization. Subsequent sections deal with four discrete steps in the LTR retrotransposon life cycle, namely, (i) transcription and transcriptional regulation; (ii) protein expression, processing, and virus-like particle assembly; (iii) reverse transcription; and (iv) integration. The study of activating mutations has unveiled a complex array of both positively and negatively acting factors responsible for Ty1’s transcriptional regulation. The discussion of reverse transcription in the remainder of the chapter focuses on the native Ty1 enzyme. Negative post transcriptional regulation of Ty1 was suggested by the paradoxical observation that Ty1 mRNA is very abundant, yet mature Ty1 proteins are scarce and transposition infrequent. In the postgenomics era, S. cerevisiae is the proving ground for new technologies that are beginning to offer unprecedented insights into the complex regulatory and metabolic networks that underlie cellular biology. The development of the research tools and the accumulated knowledge that results from their use will undoubtedly greatly facilitate Ty1 and Ty5 research in the coming years.
Full text loading...
The LTR retrotransposon life cycle begins with the transcription of an insertion in the nuclear genome. The resulting mRNA is transported out of the nucleus, where it is translated to produce the two primary gene products, homologs of retroviral proteins Gag and Pol. These proteins are then proteolytically processed by an element-encoded protease. The Gag proteins assemble in the cytoplasm into a VLP. Within the VLPs are packaged the Pol gene products, namely, PR, RT, and IN. Also packaged within the particle is template mRNA and a cellular tRNA that is used to prime reverse transcription. It is within the particle that reverse transcription of the template mRNA is carried out to generate the linear cDNA. This cDNA is then transported into the nucleus as part of a preintegration complex that includes the element-encoded IN. IN inserts the cDNA into the host genome, creating a duplication of 5 bp of host DNA, and thereby completing the life cycle. The primary difference between the LTR retrotransposons and retroviruses is that the VLP does not leave the host through membrane budding and fusion, which is carried out by the retroviral envgene product.
The LTR retrotransposon life cycle begins with the transcription of an insertion in the nuclear genome. The resulting mRNA is transported out of the nucleus, where it is translated to produce the two primary gene products, homologs of retroviral proteins Gag and Pol. These proteins are then proteolytically processed by an element-encoded protease. The Gag proteins assemble in the cytoplasm into a VLP. Within the VLPs are packaged the Pol gene products, namely, PR, RT, and IN. Also packaged within the particle is template mRNA and a cellular tRNA that is used to prime reverse transcription. It is within the particle that reverse transcription of the template mRNA is carried out to generate the linear cDNA. This cDNA is then transported into the nucleus as part of a preintegration complex that includes the element-encoded IN. IN inserts the cDNA into the host genome, creating a duplication of 5 bp of host DNA, and thereby completing the life cycle. The primary difference between the LTR retrotransposons and retroviruses is that the VLP does not leave the host through membrane budding and fusion, which is carried out by the retroviral envgene product.
Genomic organization of Ty1 and Ty5. DNA sequences are indicated by the upper open boxes. LTR sequences are shown as solid triangles. The short thin lines denote the location of the PBS and polypurine tracts (PPT), which prime minus- and plus-strand DNA synthesis, respectively. Open reading frames are indicated by the open boxes below each element. Amino acid sequence domains conserved in retroviral proteins include PR, IN, RT, and RH. RNA-binding (RB) domain denotes the finger motif characteristic of nucleocapsid proteins. Ty1 and Ty5 are drawn to scale.
Genomic organization of Ty1 and Ty5. DNA sequences are indicated by the upper open boxes. LTR sequences are shown as solid triangles. The short thin lines denote the location of the PBS and polypurine tracts (PPT), which prime minus- and plus-strand DNA synthesis, respectively. Open reading frames are indicated by the open boxes below each element. Amino acid sequence domains conserved in retroviral proteins include PR, IN, RT, and RH. RNA-binding (RB) domain denotes the finger motif characteristic of nucleocapsid proteins. Ty1 and Ty5 are drawn to scale.
A single-step selection strategy for reverse transcription. Additional details are provided in the text. The squiggly line indicates mRNA. AI, artificial intron; SD, splice donor; SA, splice acceptor.
A single-step selection strategy for reverse transcription. Additional details are provided in the text. The squiggly line indicates mRNA. AI, artificial intron; SD, splice donor; SA, splice acceptor.
Sequences that mediate Ty transcription. The arrowheads indicate the transcription start site, and the lollipop marks the site of transcription termination for Ty1. These sites define the U5, R, and U3 regions of the LTR. Other Ty1 sequences important for transcription include the TATA box and TS1 and TS2, the latter of which are required for 3′-end formation. The locations within the elements of cis-acting sequences involved in transcriptional regulation are noted: FRE, domain II, and PRE. Above the elements are listed the proteins or protein complexes that act on these sites or in their vicinity. The figures are drawn proportionally.
Sequences that mediate Ty transcription. The arrowheads indicate the transcription start site, and the lollipop marks the site of transcription termination for Ty1. These sites define the U5, R, and U3 regions of the LTR. Other Ty1 sequences important for transcription include the TATA box and TS1 and TS2, the latter of which are required for 3′-end formation. The locations within the elements of cis-acting sequences involved in transcriptional regulation are noted: FRE, domain II, and PRE. Above the elements are listed the proteins or protein complexes that act on these sites or in their vicinity. The figures are drawn proportionally.
The pheromone response and filamentous growth signaling pathways. Both pathways share several common components. Fus3p is the mating MAPK and Kss1p is the filamentous growth MAPK. The pathways culminate in the activation of Ste12p, which induces transcription of target genes. Ste12p binds to PRE in the regulatory regions of genes involved in the mating response. In the case of the filamentous growth pathway, Ste12p binds in conjunction with Tec1p to FREs. In the absence of Fus3p, Kss1p can cross-activate transcription of genes with FREs, including Ty1 elements (indicated by the dashed arrow).
The pheromone response and filamentous growth signaling pathways. Both pathways share several common components. Fus3p is the mating MAPK and Kss1p is the filamentous growth MAPK. The pathways culminate in the activation of Ste12p, which induces transcription of target genes. Ste12p binds to PRE in the regulatory regions of genes involved in the mating response. In the case of the filamentous growth pathway, Ste12p binds in conjunction with Tec1p to FREs. In the absence of Fus3p, Kss1p can cross-activate transcription of genes with FREs, including Ty1 elements (indicated by the dashed arrow).
+1 translational frameshifting as carried out by Ty1. The mRNA sequence is shown at the top of the figure. Below are the paired aminoacylated tRNAs. Circles represent the amino acid residues.
+1 translational frameshifting as carried out by Ty1. The mRNA sequence is shown at the top of the figure. Below are the paired aminoacylated tRNAs. Circles represent the amino acid residues.
Scheme for Ty1 and Ty5 protein processing. Question marks indicate predicted Ty5 proteins that have not been identified experimentally. The figure is adapted from references 86 and 101 .
Scheme for Ty1 and Ty5 protein processing. Question marks indicate predicted Ty5 proteins that have not been identified experimentally. The figure is adapted from references 86 and 101 .
Ty1 VLPs. (A) An electron micrograph of an S. cerevisiae cell overexpressing Ty1. (B) Cryoelectron microscopy three-dimensional reconstructions of Ty1 1-381 virions with icosahedral T numbers of 3 and 4 (reprinted from the Journal of Molecular Biology[ 3 ] with permission provided by H. Saibil and the publisher).
Ty1 VLPs. (A) An electron micrograph of an S. cerevisiae cell overexpressing Ty1. (B) Cryoelectron microscopy three-dimensional reconstructions of Ty1 1-381 virions with icosahedral T numbers of 3 and 4 (reprinted from the Journal of Molecular Biology[ 3 ] with permission provided by H. Saibil and the publisher).
The mechanism of reverse transcription. Details of each step are provided in the text. The template mRNA is depicted as a squiggly line. Solid lines indicate DNA; solid lines with arrowheads indicate DNA strands in the process of being extended by reverse transcriptase.
The mechanism of reverse transcription. Details of each step are provided in the text. The template mRNA is depicted as a squiggly line. Solid lines indicate DNA; solid lines with arrowheads indicate DNA strands in the process of being extended by reverse transcriptase.
Conserved features of RT. The open boxes represent the amino acid sequences of RT and RH for HIV, Ty1, and Ty5. The seven conserved amino acid sequence domains of RT are numbered and shaded in gray (60). The conserved sequence domains correspond to structural features in the enzyme that form a handlike structure: F, finger domain; P, palm; T, thumb. Note that the N terminus of Ty5 RT has not been determined. The black boxes in RH indicate conserved regions that can be aligned to the E. coli RH sequence. Conserved catalytic residues are indicated, and the amino acid positions are provided for HIV and Ty1
Conserved features of RT. The open boxes represent the amino acid sequences of RT and RH for HIV, Ty1, and Ty5. The seven conserved amino acid sequence domains of RT are numbered and shaded in gray (60). The conserved sequence domains correspond to structural features in the enzyme that form a handlike structure: F, finger domain; P, palm; T, thumb. Note that the N terminus of Ty5 RT has not been determined. The black boxes in RH indicate conserved regions that can be aligned to the E. coli RH sequence. Conserved catalytic residues are indicated, and the amino acid positions are provided for HIV and Ty1
The minus-strand primer for reverse transcription. At the top is shown the location of sequences near the Ty1 5′-LTR that are complementary to the initiator methionine tRNA. Asterisks specify residues within the initiator tRNA that are complementary to box 0, 1, and 2.1/2/2 in the Ty1 message. Note that box 0 allows for G:U pairing with the initiator tRNA. Filled circles represent sequences complementary to the Ty1 and Ty5 PBSs.
The minus-strand primer for reverse transcription. At the top is shown the location of sequences near the Ty1 5′-LTR that are complementary to the initiator methionine tRNA. Asterisks specify residues within the initiator tRNA that are complementary to box 0, 1, and 2.1/2/2 in the Ty1 message. Note that box 0 allows for G:U pairing with the initiator tRNA. Filled circles represent sequences complementary to the Ty1 and Ty5 PBSs.
Plus-strand DNA synthesis. The lowercase letters at the left of the figure refer to steps in reverse transcription depicted in Fig. 9 . Lowercase letters followed by a number indicate steps in Fig. 9 that have been expanded to include additional detail. The figures are adapted from reference 129 . (A) Model for inheritance of primer tRNA sequences. The open circle represents a mutation in the primer tRNA. The closed circle represents the first modified base, the site at which reverse transcription stops. If the mutation is copied into the plus-strand strong-stop DNA (step g), then following plus-strand transfer (step h), a heteroduplex is formed. Following mismatch repair, which is unbiased in yeast, genetic information should flow from the tRNA into the preintegrative cDNA. (B) The plus-strand primer recycling model. Details for each step are provided in the text.
Plus-strand DNA synthesis. The lowercase letters at the left of the figure refer to steps in reverse transcription depicted in Fig. 9 . Lowercase letters followed by a number indicate steps in Fig. 9 that have been expanded to include additional detail. The figures are adapted from reference 129 . (A) Model for inheritance of primer tRNA sequences. The open circle represents a mutation in the primer tRNA. The closed circle represents the first modified base, the site at which reverse transcription stops. If the mutation is copied into the plus-strand strong-stop DNA (step g), then following plus-strand transfer (step h), a heteroduplex is formed. Following mismatch repair, which is unbiased in yeast, genetic information should flow from the tRNA into the preintegrative cDNA. (B) The plus-strand primer recycling model. Details for each step are provided in the text.
The integration reaction. The conserved LTR end sequences are shown. Details of the reaction are provided in the text. TSD, target site duplication. The figure was adapted from reference 109 .
The integration reaction. The conserved LTR end sequences are shown. Details of the reaction are provided in the text. TSD, target site duplication. The figure was adapted from reference 109 .
Conserved features of IN. The open boxes represent the amino acid sequences of IN for HIV, Ty1, and Ty5. Conserved amino acid residues are indicated that define the zinc-binding motif and the catalytic domain (shaded gray). The large C termini of both Ty1 and Ty5 are rich in serine and proline. NLS, the Ty1 nuclear localization signal; TD, the Ty5 targeting domain.
Conserved features of IN. The open boxes represent the amino acid sequences of IN for HIV, Ty1, and Ty5. Conserved amino acid residues are indicated that define the zinc-binding motif and the catalytic domain (shaded gray). The large C termini of both Ty1 and Ty5 are rich in serine and proline. NLS, the Ty1 nuclear localization signal; TD, the Ty5 targeting domain.
Model for Ty1 and Ty5 target specificity. The Ty1 preintegration complex recognizes sites of RNAP III transcription, such as tRNA genes. Ty1 integrates preferentially into an approximately 750-base window upstream of the transcription start site. Ty5 integrates preferentially into an approximately 3-kb window flanking the HM silencers or the subtelomeric X repeat. Both sites are bound by the Sir complex, which includes Sir3p and Sir4p. A targeting domain (TD) is located within the C terminus of Ty5 IN that interacts with Sir4p. This interaction is thought to tether the preintegration complex to target sites.
Model for Ty1 and Ty5 target specificity. The Ty1 preintegration complex recognizes sites of RNAP III transcription, such as tRNA genes. Ty1 integrates preferentially into an approximately 750-base window upstream of the transcription start site. Ty5 integrates preferentially into an approximately 3-kb window flanking the HM silencers or the subtelomeric X repeat. Both sites are bound by the Sir complex, which includes Sir3p and Sir4p. A targeting domain (TD) is located within the C terminus of Ty5 IN that interacts with Sir4p. This interaction is thought to tether the preintegration complex to target sites.
Ty1-encoded protein nomenclature a
Ty1-encoded protein nomenclature a