Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin

Abstract

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5' mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.

Fig 1. Identification of proteins associated with the large subunit of the VLE RNAP (K2ORF6p).

The gel shows Coomassie stained proteins affinity-purified with GFP-Trap_A from strains IFO1267 (control) and IFO1267_pRKL2-4 (containing yEGFP3-K2ORF6p). Proteins identified by mass spectrometry are indicated with arrows on the right side, and also listed in Table 1. M, protein molecular mass marker (PageRuler Prestained Protein Ladder, Fermentas); the respective molecular mass values are indicated on the left side.

Fig 2. Interactions between RNAP subunits, mRNA capping enzyme, and putative helicase of the yeast VLEs.

(A) Western blot of immunoprecipitated (α-GFP IP) and mock immunoprecipitated (mock IP) proteins from IFO1267_pRKL2-5 (yEGFP3-K2ORF6p, K2ORF7p-FLAG) or control IFO1267_pRKL2-15 cells (K2ORF7p-FLAG), respectively. The strains used are indicated above the lanes. The antibodies used for Western blot are indicated on the left hand side of the strips. The proteins detected are indicated on the right hand side of the strips. Positions of the identified proteins corresponded with theoretical molecular weight of the full length recombinant proteins, as determined by positions of the protein mass markers. Input represented approximately 1/100 of the sample and IP represented approximately 1/2 of the sample in this and the other immunoprecipitation experiments. Mock immunoprecipitations in all experiments were done using empty agarose beads. The same experimental scheme is used throughout this figure. (B) Western blot analysis of immunoprecipitations from lysates from IFO1267_pRKL2-7 (yEGFP3-K2ORF6p, HA-K2ORF4p) and control IFO1267_pRKL2-13 (HA-K2ORF4p) cells (indicated above the lanes). The (α-GFP) and anti-HA (α-HA) antibodies used are indicated on the left hand side; the detected proteins on the right hand side. (C) Western blot analysis of immunoprecipitations from lysates from strains IFO1267_pRKL2-6 (yEGFP3-K2ORF6p, K2ORF3p-HA), IFO1267_pRKL2-10 (yEGFP3-K2ORF4p, K2ORF3p-HA), and IFO1267_pRKL2-14 (control). (D) Western blot analysis of immunoprecipitations from lysates from IFO1267_pRKL2-9 cells (yEGFP3-K2ORF4p, HA-K2ORF6p). (E) Western blot analysis of immunoprecipitations from lysates from IFO1267_pRKL1-4/2-4 cells (yEGFP3-K2ORF6p, K1ORF4p-HA).

Fig 3. Physical association of the putative helicase (K2ORF4p), mRNA capping enzyme (K2ORF3p), and the large RNAP subunit (K2ORF6p) of the yeast VLEs with VLE-specific DNA.

(A) Western blot of HA-K2ORF4p that was affinity-purified from lysates of IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. The strains used are indicated above the lanes. The antibody used is indicated on the left hand side of the strip. The protein detected is indicated on the right hand side of the strip. (B) PCR analysis of the presence of chromosomal (ACT, HGT1) or VLE (K1ORF3, K2ORF3) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-13 (HA-K2ORF4p) and IFO1267 (control) cells. Samples of individually performed gene-specific PCRs were analysed in 2.5% agarose gel stained with ethidium bromide. The identity of the bands (genes) is indicated on the right. M, DNA molecular mass marker (GeneRuler 100 bp Plus DNA Ladder, Fermentas). The respective values are indicated on the left. (C) Western blot of K2ORF3p-HA that was affinity-purified from lysates of IFO1267_pRKL2-14 (K2ORF3p-HA) and IFO1267 (control) cells. (D) PCR analysis of the presence of chromosomal (ACT, HGT1) or VLE (K2ORF3, K2ORF6) DNA in chromatin immunoprecipitated using anti-HA HA-7 agarose from IFO1267_pRKL2-14 and IFO1267 cells. (E) Western blot of yEGFP3-K2ORF6p that was affinity-purified from lysates of IFO1267_pRKL2-4 (yEGFP3-K2ORF6p) and IFO1267 (control) cells. (F) PCR analysis of the presence of chromosomal (ACT, HGT1) or VLE (K2ORF3, K2ORF6) DNA in chromatin immunoprecipitated using GFP-Trap agarose beads from IFO1267_pRKL2-4 and IFO1267 cells.

Fig 4. Promoters of yeast VLEs contain initiator region (INR) responsible for non-templated 5′ polyadenylation of mRNAs.

(A) Sequence logo of the initiator region in promoter sequences of Vaccinia virus intermediate genes [41]. (B) Sequence logo of the initiator region in promoter sequences of Vaccinia virus late genes [41]. (C) Sequence logo of the putative INR identified in promoters of 12 ORFs encoded by pGKL elements. (D) 5′ RACE-PCR analysis of the G418^R gene from the IFO1267_pRKL1-1 strain. In this and the following panels, the upper sequence corresponds to the template (plasmid) DNA and the UCS is indicated; sequences situated below represent individual sequenced cDNA clones (the 5′ untranslated region is shown in full till the translation start codon, ATG). Guanosine residues corresponding to the original 5′ mRNA caps which were present in some of the cDNA clones are omitted in this representation for clarity. (E) 5′ RACE-PCR analysis of the G418^R gene from the IFO1267_pRKL1-2 strain bearing a promoter mutation in the putative INR reducing the number of consecutive adenosine residues in the template. (F) 5′ RACE-PCR analysis of the G418^R gene from the IFO1267_pRKL1-3 strain bearing promoter mutations in the putative INR abolishing consecutive adenosine residues in the template.

Fig 5. RNA stem loop structures influence the 3′ end formation of VLE-specific mRNAs in vivo.

(A) Schematic representation of recombinant pGKL1 elements where the G418^R gene is followed by the coding sequence of wild-type (pRKL1-5) or modified (pRKL1-6, pRKL1-7) 3′ untranslated region of the K2ORF5 gene. TIR—terminal inverted repeat. (B) 3′ RACE-PCR analysis of individual mRNAs corresponding to the G418^R gene expressed from modified pGKL1 elements. Samples were analyzed in 3.0% agarose gel stained by ethidium bromide. The strains used to purify the RNA are indicated above the lanes. M, DNA molecular mass marker (GeneRuler 100 bp Plus DNA Ladder, Fermentas). The respective values are indicated on the left. Specific products that were cloned to the pCR4-TOPO vector and used for sequencing are labelled with asterisks. Reverse transcription was carried out in the presence (+RT) and absence (-RT) of reverse transcriptase. (C) 3′ RACE-PCR analysis of the G418^R gene from IFO1267_pRKL1-5 strain. In this and the following panels, the upper sequences on the right correspond to the template (plasmid) DNA; sequences situated below represent 3′ end regions of individual sequenced cDNA clones. Positions of the putative RNA stem loops are indicated above the sequences. Predicted RNA stem loops are displayed as cDNA nucleotide letters in circles on the left and the values of Gibbs free energy (ΔG) in kcal/mol are displayed for each structure. Stem loop distances from the gene stop codon are shown as numbers of nucleotides. The last few 3′ end nucleotides of the experimentally determined 3′ ends of cDNA are shown as letters enlarged proportionally to their occurrence (in %) in the sequenced clones in the case when these nucleotides were detected in at least two independent clones. (D) 3′ RACE-PCR analysis of the G418^R gene from the IFO1267_pRKL1-6 strain. Mut, the mutated stem loop. (E) 3′ RACE-PCR analysis of the G418^R gene from IFO1267_pRKL1-7 strain. Mut, the mutated stem loop; Res, the rescued stem loop.

Fig 6. Predicted 3D structure of yeast VLE non-canonical RNAP encoded by the ORF6 and ORF7 genes.

(A) Schematic representation of the primary sequence of pGKL-encoded RNAP showing similarity to conserved regions of the catalytic subunits of canonical multisubunit RNAPs. β subunit conserved regions (blue) and β′ subunit conserved regions (red) present in K2ORF6p and K2ORF7p are drawn to scale. Conserved regions are named according to ref. [46]. Sequence alignments for newly detected similarity of ORF6 protein products to βa1, βa6, βa13 and βa16 conserved regions are provided in S6 Fig. BH, Bridge helix; FL2, Fork loop 2; Sw2, Switch 2; Sw3, Switch 3; Sw5, Switch 5; TH1, Trigger helix 1; TH2, Trigger helix 2; TL, Trigger loop. (B) 3D crystal structure of Saccharomyces cerevisiae RNA polymerase II elongation complex showing the Rpb2 subunit (β subunit homolog, light blue), DNA (green) and RNA (cyan). This figure is based on 2NVQ. (C) 3D model of pGKL RNAP showing K2ORF6p residues 1–693 (β subunit homolog, blue), DNA (green) and RNA (cyan). Nucleic acids in this as well as the following structures are based on 2NVQ. The structural similarity between the pGKL RNAP β subunit and RNAP II Rpb2 as calculated by DaliLite v. 3 is a root-mean-square deviation (RMSD) of 3.9 Å over 655 aligned Cα positions, 23% sequence identity, and the TM-score 0.87. (D) 3D crystal structure of Saccharomyces cerevisiae RNAP II elongation complex showing the Rpb1 subunit (β′ subunit homolog, pink), DNA (green) and RNA (cyan). Arrows indicate β′ regions shared by multisubunit RNAPs (red) that are clearly missing in the VLE RNAP. This figure is based on 2NVQ. (E) 3D model of pGKL RNAP showing K2ORF6p residues 754–882 and 894–974 (β′ subunit homolog, red), K2ORF7p residues 1–52 and 103–132 (β′ subunit homolog, orange), DNA (green) and RNA (cyan). The structural similarity between pGKL RNAP β′ subunit and RNAP II Rpb1 as calculated by DaliLite v. 3 is a RMSD of 2.3 Å over 123 aligned Cα positions, 31% sequence identity, and the TM-score 0.89 for K2ORF6p residues 754–882; RMSD of 1.4 Å over 80 aligned Cα positions, 21% sequence identity, and the TM-score 0.86 for K2ORF6p residues 894–974; RMSD of 2.9 Å over 43 aligned Cα positions, 23% sequence identity, and the TM-score 0.57 for K2ORF7p residues 1–52; RMSD of 1.3 Å over 29 aligned Cα positions, 13% sequence identity, and the TM-score 0.56 for K2ORF7p residues 103–132. For details concerning structure modelling see Materials and methods.

Fig 7. Phylogenetic analysis of yeast VLE RNA polymerases with canonical RNAPs.

A phylogram of β′ subunit homologs based on amino acid sequence alignment of β′ subunit conserved regions of selected canonical RNAPs and those β′ subunit conserved regions present in ORF6 and ORF7 genes of the yeast VLEs. The maximum likelihood unconstrained tree is displayed as an unrooted phylogram where the length of the branches is proportional to the calculated evolutionary distance of individual sequences. Leaves defining the different classes of RNAPs are labeled. Used abbreviations for RNAP groups: aRNAP, archaeal RNAP; bRNAP, bacterial RNAP; eRNAP, eukaryotic RNAP; pRNAP, plastid RNAP. Length scale of branches of an average value of 0.5 substitution per amino acid residue is shown as a line near the tree. Selected branch support values calculated from 1 000 ultrafast bootstrap replicates optimized using nearest neighbour interchange (NNI) to reduce overestimating support are indicated in black. For details concerning phylogenetic analysis see Materials and methods.

Fig 8. Upstream promoter elements of yeast VLEs show high sequence similarity to promoters of poxviral early genes.

(A) Vaccinia virus early promoter consensus motif termed upstream control element (UCE) calculated from 84 sequences identified from genome-wide RNA-sequencing experiments in ref. [48]. (B) Graph showing the number of UCE sequences as a function of their distances to the transcription start sites (TSS) of 84 ORFs as annotated in ref. [48]. (C) Extended promoter consensus motif of pGKL elements preceding 15 ORFs. This motif contains the upstream conserved sequence (UCS) which is universal among yeast VLEs. (D) Graph showing the number of extended UCS sequences as a function of their distances to the TSS of 15 pGKL-encoded ORFs as determined in 5′ RACE-PCR experiments. For promoters with a putative initiator region the first adenosine residue in the region was considered to be the TSS. For more information concerning promoter characterization see Materials and methods.

Fig 9. Model of the yeast VLE transcription initiation and termination.

(A) Putative helicase K2ORF4p (ORF4p, yellow) binds to the VLE DNA, presumably to the upstream conserved sequence (UCS, black) which is related to the early promoter element of poxviruses. (B) K2ORF4p recruits the RNAP complex (ORF6p/ORF7p, brown) to the transcription initiation site, which usually contains the initiator region (INR, grey) responsible for RNAP slippage and subsequent 5′ mRNA polyadenylation. (C) ATP hydrolysis by K2ORF4p releases it from the transcription preinitiation complex to allow RNAP to escape from the initiation site and produce mRNA (RNA, green) containing a 5′ end poly(A) leader. This RNA can be subsequently 5′ capped by the K2ORF3p viral-like mRNA capping enzyme (ORF3p, orange). Transcription termination most likely proceeds in a factor-independent manner that involves intrinsic terminators consisting of RNA stem loop structure(s) and 3′ terminal U-tract.