Point mutations in the Rpb9-homologous domain of Rpc11 that impair transcription termination by RNA polymerase III

Abstract

RNA polymerase III recognizes and pauses at its terminator, an oligo(dT) tract in non-template DNA, terminates 3' oligo(rU) synthesis within this sequence, and releases the RNA. The pol III subunit Rpc11p (C11) mediates RNA 3'-5' cleavage in the catalytic center of pol III during pausing. The amino and carboxyl regions of C11 are homologous to domains of the pol II subunit Rpb9p, and the pol II elongation and RNA cleavage factor, TFIIS, respectively. We isolated C11 mutants from Schizosaccharomyces pombe that cause pol III to readthrough terminators in vivo. Mutant RNA confirmed the presence of terminator readthrough transcripts. A predominant mutation site, F32, resides in the C11 Rpb9-like domain. Another mutagenic approach confirmed the F32 mutation and also isolated I34 and Y30 mutants. Modeling Y30, F32 and I34 of C11 in available cryoEM pol III structures predicts a hydrophobic patch that may interface with C53/37. Another termination mutant, Rpc2-T455I, appears to reside internally, near the RNA-DNA hybrid. We show that the Rpb9 and TFIIS homologous mutants of C11 reflect distinct activities, that differentially affect terminator recognition and RNA 3' cleavage. We propose that these C11 domains integrate action at the upper jaw and center of pol III during termination.

Figure 1.

Schematic representations of the tRNA-mediated suppression (TMS) systems, that use (A) monomeric and (B) dimeric tRNA reporter genes, the latter designated pDRT. The complementary read through (cRT) region in the monomeric construct is highly complementary to the sequence of the tRNA, so that if synthesized, it can fold back and interfere with processing of the tRNA (33). Note that for B, the upstream tRNA provides the pol III upstream promoter element (37). (C) Relative TMS activity of dimeric (pDRT) constructs bearing oligo(dT) tracts of varying length, T2-T7, in the T1 region, assayed in strain yAS99 as described (33) (actual activity of pDRT-4T was ∼75%). pJK148 is a negative control that contains no suppressor tRNA gene.

Figure 2.

S. pombe C11-mutants isolated from a library of PCR-mutagenized C11 (28), using the pDRT-6T dimeric tRNA reporter gene. (A) Sequence alignment of C11 and the three unique C11-mutants numbered 49, 51 and 111. Asterisks indicate invariant cysteines in the Zn-binding motifs. (B) tRNA-mediated suppression phenotypes of the C11-mutants 49, 51 and 111 shown in A and vector control as indicated; red colony color reflects no suppression.

Figure 3.

(A) Sequence alignment of the N-terminal regions of C11 and Rpb9p, above and below horizontal line, respectively, from several organisms. Invariant Cysteines are indicated by asterisks below the sequences. Highly conserved residues are indicated by downward arrows above the sequences, and for S. cerevisiae Rpb9 these are numbered below the sequence. The C11 F32 position is indicated by an asterisk above. (B) Sequence alignment of homologous regions of Rpb2p and Rpc2p from several organisms, above and below horizontal line, respectively. The asterisk indicates position D-294 (S. cerevisiae numbering). The gray line above the sequences spans the region that when deleted in S. pombe Rpc2p leads to loss of C11 (31). The asterisks below the alignment indicate positions of mutations in S. cerevisiae RET1 (Rpc2) that cause decreased termination (18). C) Structure view of a segment of the yeast pol II core (PDB ID 3M40) that includes the N-terminal domain of Rpb9p shown as yellow ribbon. The highly conserved F, L andY highlighted in A above as well as F4 are represented as side chains with space-filling dots. Side chains of L298 and L311 of Rpb2 are colored white with space-filling dots. The backbone positions and side chains of the clustered cysteines in Rpb9 are shown in cyan, and the coordinated Zn as a purple sphere. (D) Structure model (PDB ID 3M40) showing the Rpb9p N-12 side chain, which is highly conserved in C11, indicated by a yellow arrow. The side chain of the highly conserved D-294 in Rpb2p is indicated by an asterisk; the N12-D94 distance is <4 Å. The region of Rpb2 that corresponds to the deletion in S. pombe Rpc2p that leads to loss of C11 (31) is colored black. (E) TMS phenotypes of the site-directed mutants, C11-F32S and C11-C25R, F32SC11, as well as wild-type C11 and vector control (see text). (F) Coimmunoprecipitation of HA-tagged Rpc11p-WT and -F32S with Flag-Rpc2p. Strain yYH3272 carrying Flag-Rpc2p (37) was transformed with HA-C11-WT or HA-C11-F32S, and extracts made. Inputs (lanes 1 and 2), the products of mock IP using nonimmune IgG (lanes 3 and 4), and products of IP using anti-Flag IgG were blotted and probed with anti-HA Ab. The lanes between 4 and 5, and 5 and 6, were not loaded.

Figure 4.

Side-by-side analyses in yYH1 and yJI1 of multiple S. pombe pol III mutants. Strains yYH1 (A) and yJI1 (B) bear monomeric and dimeric suppressor tRNA genes, respectively. The expression plasmids carrying the alleles to be tested (C11, Rpc2, etc.) were assayed in both strains and displayed according to the schematic to the right. (C) Northern blot of RNAs isolated from yYH1, which carries a reporter tRNA gene with a dT(7) terminator, after transformation with the plasmid-borne expression alleles indicated above lanes 3–8; lanes 1 and 2 contain RNA from positive and negative control strains. Upper panel shows blot probed for the readthrough transcript from the suppressor tRNA dT(7) gene. Lower panel shows same blot probed for U5 snRNA as a loading control. Lane 1 was intentionally under loaded so that the intensity of the yAS68 RT band would be closer to the linear range of detection, and to avoid contamination of other lanes. (D) Northern blot of RNAs isolated from yAS77, which carries a reporter tRNA gene with a dT(6) terminator; lanes are as in C above. Quantitation of C and D is reported in Table 2.

Figure 5.

Modeling termination-relevant mutations in the pol III cyroEM structure. (A and B) Views of pol II in which the surface of Rpb9 N-terminal domain is yellow with the Glu-36 position that corresponds to C11-F32 colored red (PDB ID 3M40). (C) The cryoEM structure (EMD-1802) (32) was fit with the core pol II crystal structure (PDB 1WCM) (52) lacking the stalk and the C-terminal domain of Rpb9p. Fit was by manual manipulation followed by automated fit using UCSF Chimera (35) (final lowest average value = 0.9628 for 23833 atoms, 4042 outside contour). The view is similar to Figure 2A and B in (2) and the front view in Figure 2B in (32). Pol II is depicted as ribbon except for the Rpb9 N-terminal domain which is a yellow surface view, with Glu-36 (corresponding to C11-F32) colored red and indicated by an orange arrow. Rpb2p is colored light green except for two regions proximal to the C53/37 density, which are cyan and red, indicated by dashed arrows. Cyan corresponds to a region in S. pombe Rpc2 that when deleted causes loss of C11 (31), and the red helix corresponds to the region containing RET1 decreased termination mutations indicated in Fig. 3B. Rpb1 is colored blue. Envelope densities corresponding to the protrusion, stalk and foot, and C53/37 are indicated (32). (D) Alignment of sequences from several organisms of a central homologous region of Rpb2 and Rpc2. Blue asterisk above the sequences indicates S480 in Rpb2 and T455 in Rpc2, the latter when mutated as part of this study led to decreased termination. The red asterisk indicates Rpb2 position Q481 (see text). Pink asterisks below Rpc2 indicate mutations in RET1 that decrease termination (18). (E–G) Pol II (PDB ID 3M40) with an RNA–DNA hybrid; RNA backbone is red. Rpb2 S480 and Q481 are shown as blue and red spheres. RET1 mutations indicated by asterisks in B are shown as pink spheres. The N-terminal domain of Rpb9 is shown as yellow surface. In F and G, the sequence tract corresponding to the region in spRpc2 that when deleted causes loss of C11 is colored black and the region corresponding to the 300–325 regions of RET1 (see Figure 3B) is colored blue (31).

Figure 6.

Codon randomization leads to additional C11 termination readthrough mutants. (A) TMS spot assay comparing suppression activities of various individual position specific mutations of C11. See Table 3 for complete list. (B) TMS spot assay showing activities of mutants isolated by screening using the mini-library containing a mixture of all codon randomizations of 12 codons (see text). The identity key for the three columns is to the right. See Table 4 for complete list. (C) Pol II homology model with the three most frequent C11 mutants in red, each indicated by an asterisk, as reflected on the Rpb9p surface (yellow) as in Figure 5A.