Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

Figures

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  Figure 1. Schematic of the Ty1 element DNA, the Ty1 RNA 5′ TIPIRT domain and in vitro transcripts analyzed by SHAPE chemistry. Ty1 retrotransposon DNA consists of two 334 bp long terminal repeats (LTRs; represented by tripartite rectangles) composed of U3 (unique to the 3′ end of the RNA), R (repeated at the 5’ and 3′ ends of the RNA) and U5 (unique to the 5’ end of Ty1 RNA). LTRs flank a central coding region (black bar). The GAG and POL ORFs are denoted by rectangles above the element. Below the DNA, the 5′ leader of Ty1 RNA from nucleotide 1 (the beginning of “R”) to nucleotide 448 (in the GAG ORF), which includes the TIPIRT domain (nucleotides 1–380), is represented below the Ty1 element DNA. Vertical white rectangles denote sequences that are essential for initiation of reverse transcription (1/7 and 264/270 pseudoknot S1 stem; 95/104-PBS; 110/116-Box 0; 144/149-Box 1; 155/168-CYC5, including Box 2.1). The horizontal white rectangle spanning nucleotide 237–380 denotes a region required for Ty1 RNA packaging. The schematic at the bottom represents the in vitro transcript (nucleotides 1–513, plus an FTL tag indicated by the striped box) that was analyzed by SHAPE. Grey shading…

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  Figure 2. SHAPE reactivities and secondary structure model of the 5′ leader of Ty1 RNA. Nucleotides are colored according to their SHAPE reactivities, which are indicated on the color bar at the bottom left. Regions of low reactivity have a high probability of being constrained within secondary or tertiary structure. The AUG nucleotides shaded in grey comprise the start codon of GAG. The pseudoknot core contains stem S1 (blue shading), loop L2 (nucleotide 263, yellow shading), and stem S2 (green shading). Pseudoknot loops L1 (nucleotides 9–254) and L3 (nucleotides 271–318) are not shaded. The SL1a hairpin (pink shading) and SL3a hairpin (purple shading) are indicated.

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  Figure 3. Model of a symmetrical Ty1 RNA kissing complex containing two Ty1 RNA pseudoknots interacting via two 7-base-pair intermolecular RNA duplexes formed between apical motifs in stem-loop SL1a (pink arc) and SL3a (orange arc). The pseudoknot stems are shaded in blue.

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  Figure 4. Relative evolutionary conservation of each nucleotide overlayed on the secondary structure model of the 5′ leader of Ty1 RNA. The color of each RNA base indicates its degree among conservation among 102 Ty1 and Ty2 elements from the genus Saccharomyces. Categories of conservation are as follows: red, 100% conserved among 102 Ty1 and Ty2 elements in the genus Saccharomyces; purple, 100% conserved in 66 Saccharomyces cerevisiae Ty1 elements; grey, not 100% conserved in either set.

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  Figure 5. Assay for helper-mediated retrotransposition of mini-Ty1his3AI. A complete Ty1 element is shown at the top for reference. The mini-Ty1his3AI element and helper-Ty1 element are each expressed from the GAL1 promoter (labeled rectangle), which is fused to the transcription start site of Ty1-H3 at the first nucleotide of the R domain in the 5′ LTR. GAL1:mini-Ty1his3AI is carried on a URA3-based plasmid and GAL1:helper-Ty1 is contained on a TRP1-based plasmid (not illustrated). The elements are co-expressed in an spt3∆ strain lacking endogenous Ty1 element transcription. The internally deleted mini-Ty1his3AI element contains 5′ sequences corresponding to nucleotides 1–575 of Ty1 RNA, as well as the last 357 nucleotides of Ty1, including the 3′ polypurine tract (not illustrated) and 3′ LTR. The his3AI retrotransposition indicator gene, consisting of the HIS3 marker gene interrupted by an antisense intron (boxed arrowhead), is inserted in the mini-Ty1 between the 5′ leader and 3′ LTR. The direction of mini-Ty1his3AI transcription from the GAL1 promoter (denoted by the arrow atop the GAL1 rectangle) is opposite to the direction of his3AI transcription (denoted by an arrow atop the HIS3…

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  Figure 6. Retrotransposition of mini-Ty1his3AI elements with mutations in the Ty1 pseudoknot core. The schematic (top left) shows the secondary structure of the Ty1 pseudoknot core and portions of the L1 and L3 loops. Blue shading, stem S1; yellow shading, loop L2; green shading, stem S2; pink shading, SL1a hairpin, a segment of the L1 loop; orange shading, SL3a hairpin, a portion of the L3 loop. Dotted lines represent bases in loops L1 and L3 that are not shown. Labeled, boxed schematics show the nucleotide substitutions or additions in each mutant mini-Ty1his3AI element analyzed. Black letters represent wild-type nucleotides; red letters represent nucleotide substitutions or additions; and green letters represent compensatory substitutions that restore base-pairing with nucleotide substitutions. The percentage below each box is the median frequency of helper-mediated retrotransposition of the mini-Ty1his3AI bearing the indicated mutation divided by the median helper-mediated retrotransposition frequency of the mini-Ty1his3AI element with wild-type Ty1-H3 sequence, +/− the 95% confidence interval.

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  Figure 7. Retrotransposition of mini-Ty1his3AI elements with mutations in stem-loops SL1a and SL3a. The schematic (top) shows the secondary structure of the Ty1 pseudoknot core and loops L1, L2 and L3, with the SL1a hairpin (pink shading) and SL3a hairpin (purple shading) highlighted. A second schematic (second from top, left) shows the proposed kissing loop interaction between the seven apical nucleotides of hairpin SL1a (pink shading) and seven apical sequences of the SL3a hairpin. Labeled, boxed schematics show the nucleotide substitutions or additions in each mutant mini-Ty1his3AI element analyzed. Black letters indicate wild-type sequence; red letters indicated nucleotide substitutions or additions; and green letters indicate compensatory substitutions that are predicted to restore base-pairing with nucleotide substitutions. The percentage below each box is the median frequency of helper-mediated retrotransposition of each mini-Ty1his3AI bearing the indicated mutation divided by the median helper-mediated retrotransposition frequency of the mini-Ty1his3AI element with wild-type Ty1-H3 sequence, +/− the 95% confidence interval.

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  Figure 8. Levels of mini-Ty1his3AI RNA bearing different mutations and helper-Ty1 RNA. (A) Northern blot analysis of strains carrying the pGAL1:mini-Ty1his3AI plasmid harboring wild-type Ty1 sequences (WT) or mutant Ty1 sequences and the pGAL1:helper-Ty1 induced for 24 hours in galactose-containing medium. The presence or absence of the pGAL1:helper-Ty1 plasmid is indicated by + and – symbols, respectively, above the blot. Labels for mutations correspond to those in Figure 6 and Figure 7; (B) Measurement of the median GFP activity in 10,000 cells of two different transformants of each pGAL1:GAGNT:GFP plasmid containing wild type Ty1 TIPIRT domain sequences or derivatives with mutations named as in Figure 6 and Figure 7. Strains were induced in galactose-containing medium for 2.5 h. Error bars are the standard deviation of the median GFP activity in each of two transformants.