The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination - PubMed (original) (raw)

The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination

S Chédin et al. Genes Dev. 1998.

Abstract

Budding yeast RNA polymerase III (Pol III) contains a small, essential subunit, named C11, that is conserved in humans and shows a strong homology to TFIIS. A mutant Pol III, heterocomplemented with Schizosaccharomyces pombe C11, was affected in transcription termination in vivo. A purified form of the enzyme (Pol III Delta), deprived of C11 subunit, initiated properly but ignored pause sites and was defective in termination. Remarkably, Pol III Delta lacked the intrinsic RNA cleavage activity of complete Pol III. In vitro reconstitution experiments demonstrated that Pol III RNA cleavage activity is mediated by C11. Mutagenesis in C11 of two conserved residues, which are critical for the TFIIS-dependent cleavage activity of Pol II, is lethal. Immunoelectron microscopy data suggested that C11 is localized on the mobile thumb-like stalk of the polymerase. We propose that C11 allows the enzyme to switch between an RNA elongation and RNA cleavage mode and that the essential role of the Pol III RNA cleavage activity is to remove the kinetic barriers to the termination process. The integration of TFIIS function into a specific Pol III subunit may stem from the opposite requirements of Pol III and Pol II in terms of transcript length and termination efficiency.

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Figures

Figure 1

Sequence alignment of C11 with RNA polymerase subunits and factor TFIIS. (A) Alignment of the C11 sequence with Pol I A12.2 and Pol II B12.6 subunits from the yeast Saccharomyces cerevisiae. Amino acids identical in at least two sequences are boxed. Cysteines of the two potential zinc-binding domains are indicated by stars. (B) Schematic representation of the human gene encoding HsC11p. (C) Alignment of the sequences of C11 subunits from S. cerevisiae (C11), S. pombe (SpC11p), and Homo sapiens (HsC11p). Amino acids identical in at least two sequences are boxed. Cysteines of the two potential zinc-binding domains are indicated by stars. (D) Sequence alignment of the carboxy-terminal zinc-binding domains of Pol III C11 subunit, of Pol II B12.6 subunit, and of elongation factor TFIIS from S. cerevisiae (Sc), Drosophila melanogaster (Dm), Mus musculus (Mm), and H. sapiens (Hs). Amino acids identical in at least four sequences are boxed.

Figure 2

Heterocomplemention of C11 by its S. pombe ortholog SpC11p. (A) Interspecific complementation of rpc11::HIS3 by S. pombe gene encoding SpC11p. Haploid strains harboring the rpc11::HIS3 allele were complemented by pGEN–RPC11 (constitutively expressing the C11 subunit) or pGEN–SpC11 (constitutively expressing the S. pombe SpC11p subunit). These constructions were obtained by plasmid shuffling (see Materials and Methods). Growth was tested by spotting 5 μl of different dilutions of a liquid cell culture on YPD plates and incubating at 16°C, 30°C and 37°C for 7, 3, and 5 days, respectively. (B) Pol III Δ lacks subunit C11. Wild-type Pol III (lane 1) and Pol III purified from the heterocomplemented strain (Pol III Δ, lane 2) were analyzed by electrophoresis in a 4–15% SDS–polyacrylamide gel and silver stained. (*) Two major contaminants present in the wild-type enzyme preparation; (arrowheads) positions of C11 and C53 subunits in the wild-type enzyme.

Figure 2

Figure 3

Stalled Pol III Δ incorporates an additional nucleotide and does not cleave the nascent RNA transcript. (A) Sequence of the first 22 bases of the coding strand of SUP4 gene. The size of the 17-mer transcript obtained in the absence of GTP (3 XTPs) is indicated. (B) A stable initiation complex was formed on the SUP4 gene as described in Materials and Methods. Wild-type Pol III or Pol III Δ were then added together with ATP (600 μ

), CTP (600 μ

), and [α-32P] UTP (3 μ

, 400 μCi/nmole) in the absence or in the presence of 3′-OMe-GTP (600 μM) as indicated. Reaction products were separated by electrophoresis on a 15% polyacrylamide, 7

urea gel. The position of the 17-mer, 18-mer, and 18*-mer transcripts (see text) is indicated. (C) A stable initiation complex was formed as described above. Wild-type Pol III or Pol III Δ were then added together with ATP (600 μ

), CTP (600 μ

), and [α-32P] UTP (3 μ

, 400 μCi/nmole) without recombinant C11 subunit (lanes 1,2, respectively). A 50

excess of recombinant C11 subunit was incubated for 10 min with Pol III Δ before the transcription reaction (lane 3) or added for 2 min after the transcription reaction (lane 4). Reaction products were separated by electrophoresis on a 15% polyacrylamide, 7

urea gel. The positions of the 17-mer and 18-mer, transcripts (see text) are indicated. (D) Transcript cleavage by halted ternary complexes. Wild-type Pol III or Pol III Δ ternary complexes formed in the presence of 3 XTPs were isolated on Sepharose CL-2B columns as described in Materials and Methods. Pol III Δ ternary complexes were then incubated or not for 10 min with a 50

excess of recombinant C11 subunit. Ternary complexes were then incubated in transcription buffer containing 8 m

MgCl2 in the absence of nucleotide for various periods of time. Sizes of transcripts are at left.

Figure 3

), CTP (600 μ

), and [α-32P] UTP (3 μ

, 400 μCi/nmole) in the absence or in the presence of 3′-OMe-GTP (600 μM) as indicated. Reaction products were separated by electrophoresis on a 15% polyacrylamide, 7

), CTP (600 μ

), and [α-32P] UTP (3 μ

, 400 μCi/nmole) without recombinant C11 subunit (lanes 1,2, respectively). A 50

excess of recombinant C11 subunit. Ternary complexes were then incubated in transcription buffer containing 8 m

MgCl2 in the absence of nucleotide for various periods of time. Sizes of transcripts are at left.

Figure 4

Stalled Pol III Δ ternary complexes are transcriptionally incompetent. Kinetic of elongation on the SUP4 gene by wild-type enzyme (A) or Pol III Δ (B,C). Wild-type Pol III-labeled 17-mer ternary complexes (A) or Pol III Δ-labeled 18-mer ternary complexes (B) were isolated and elongation was resumed for various periods of time as indicated, by adding unlabeled ATP, CTP, GTP (600 μ

each) and UTP (30 μ

). The size of the RNA transcripts and the four major transcriptional pause zones (P1–P4) are indicated at left. (C) Isolated Pol III Δ-labeled 18-mer ternary complexes were incubated for 10 min with a 50

excess of recombinant C11 subunit, then incubated with unlabeled nucleotides as above for 5 or 60 sec, as indicated.

Figure 5

Pol III Δ is affected in pausing and transcription termination. (A) Termination and pausing on SUP4 gene. A schematic representation of the SUP4 gene terminator with the T1 and T2 blocks is shown. Stable initiation complexes were formed on the SUP4 gene as described in Materials and Methods. Wild-type enzyme (lanes 1,3) or Pol III Δ (lanes 2,4) was then added together with ATP, CTP, GTP (600 μ

each) and [α-32P]UTP (30 μ

, 20 μCi/nmole). Reaction products were separated by electrophoresis on a 6% polyacrylamide, 7

urea gel. Lanes 3 and 4 represent a longer exposure time of lanes 1 and 2 to reveal paused transcripts. Large transcripts accumulating at the top of the gel are indicated as RNAs x. The positions of full-length transcripts terminated at T1 or T2 and RNAs corresponding to transitory paused ternary complexes (P2–P4) are indicated. (B) Pol III Δ reads through SNR6 gene terminator. Transcription was performed on a purified DNA fragment of 200 bp obtained after double digestion _Hin_dIII–_Sma_I of the pTaq6 plasmid that contains the truncated version of the yeast SNR6 gene (see Materials and Methods). The _Sma_I site is located 40 nucleotides downstream of the SNR6 gene terminator sequence. Wild-type Pol III or Pol III Δ were added together with ATP, CTP, GTP (600 μ

each) and [α-32P] UTP (30 μ

, 20 μCi/nmole) in the absence or in the presence of TFIIIB prealably assembled onto the DNA. Reaction products were separated by electrophoresis on a 6% polyacrylamide, 7

urea gel. The positions of the full-length U6 RNA (U6) and of the run-off transcript (RO) are indicated.

Figure 5

each) and [α-32P]UTP (30 μ

, 20 μCi/nmole). Reaction products were separated by electrophoresis on a 6% polyacrylamide, 7

each) and [α-32P] UTP (30 μ

, 20 μCi/nmole) in the absence or in the presence of TFIIIB prealably assembled onto the DNA. Reaction products were separated by electrophoresis on a 6% polyacrylamide, 7

urea gel. The positions of the full-length U6 RNA (U6) and of the run-off transcript (RO) are indicated.

Figure 6

C11 is involved in class III genes termination in vivo. The heterocomplemented strain that carries the ocher allele of ade2-101, was transformed with one plasmid of the pSUP4 series (URA3, CEN6) that contained the ocher suppressing allele of SUP4-o tRNATyr and 3′ deletion mutants with altered terminators. A schematic representation of the pSUP4 serie is drawn on the left. Transformants were replicated in three different media. The growth of transformants was assayed on a media lacking Ade (−ADE). Cells in which termination of the SUP4 gene was defective were unable to grow. Color determination was performed on rich medium (YPD). Cells in which no or weak suppression of the ade2-101 allele occurred gave rise to red colonies. The controls show the growth of the cells on a medium containing Ade and Ura. Growth was scored after 4 days at 30°C. Eight independent transformants exhibited the same phenotype for growth and color assays.

Figure 7

Low UTP concentrations corrects the termination defect of Pol III Δ. A stable initiation complex was formed on the SUP4 gene as described in Materials and Methods. Wild-type enzyme or Pol III Δ was then added together with ATP, CTP, and GTP (600 μ

each) and [α-32P]UTP (400 μCi/nmole) at concentrations varying between 300 and 1 μ

, as indicated. Reaction products were separated by electrophoresis on a 6% polyacrylamide, 7

urea gel. Large transcripts accumulating at the top of the gel are indicated by RNAs x. The position of full length SUP4 tRNA transcripts terminated at T1 or T2 is indicated.

Figure 8

Immunolocalization of subunit A12.2 in the Pol I three-dimensional structure. (A–C) Average images of RNA Pol I dimers labeled with anti-A12.2 antibodies were obtained by analysis of 473 molecular images. The stain-excluding protein densities are outlined by contours of equal density. The difference image between the antibody-labeled and the unlabeled molecule is shown in yellow where the contours correspond to positive differences thresholded at a significance level of 5%. Each panel corresponds to a distinct dimer view identified by the angle of the internal two-fold axis with the normal to the plane. For the 90° dimer view (A), the images were transformed to show the binding in the upper molecule, thus the antibody interaction site is identified by comparing the upper to the lower monomer. For the 30° dimer view (B,C), two independent images were calculated where either the upper (B) or the lower monomer (C) was labeled. (D) Surface representation of the three-dimensional RNA Pol I model showing the position of A12.2 in yellow. Taking the monomeric envelope in the top left panel as a reference; the top right panel represents the envelope viewed after a counterclockwise tilt of 30° around a vertical axis. The reference is viewed from the top in the bottom left panel and from the bottom in the bottom right panel. (A–C) Bar, 10 nm; (D) bar, 5.5 nm.

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The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination - PubMed (original) (raw)