The C53/C37 subcomplex of RNA polymerase III lies near the active site and participates in promoter opening

Abstract

The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3' end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF.

FIGURE 1.

Properties of the 3′-overhang DNA construct used for factor-independent formation of pol IIIΔ elongation complexes. A, shown is the sequence of the 3′-overhang template highlighting sites of GpA-primed initiation and arrest of elongation. B, stepwise walking of pol IIIΔ elongation complexes is shown, demonstrating reiterative slippage and extension of the initially transcribed sequence. Elongation complexes arrested at C21 were formed by GpA-primed initiation in the presence of ATP and CTP; unincorporated nucleotides were removed as described under “Experimental Procedures” before distribution of aliquots to tubes providing 10 mm MgCl₂ and 50 μm ATP (lane 1), UTP (lane 2), or ATP+UTP (lane 3). The anticipated lengths of transcription products are identified at the right (based on indexing to 25- and 28-nt RNA markers). The DNA recovery marker (rm) was added with the reaction stop buffer. C, incorporation of adjacent ABUMP residues arrests elongation. Pol IIIΔ elongation complexes halted at C21 were allowed to elongate to G40 in the presence of sufficient unlabeled CTP to dilute the specific activity 50-fold, 3′-OMeGTP (lanes 1–3) and 100 μm ABUTP (lane 2) or UTP (lane 3). The expected size of the 3′-OMeGTP-terminated transcript (G40) is shown at the left.

FIGURE 2.

The C53 and C11 subunits of the pol III elongation complex cross-link to ABUMP incorporated at the 3′ of the nascent transcript. A, pol IIIΔ elongation complexes formed with the template shown in Fig. 1A were arrested by incorporation of adjacent ABUMP residues and isolated by chromatography on Sephacryl S300. The peak fraction containing the nearly excluded pol IIIΔ elongation complex was distributed into tubes providing 100 nm each of untagged C11 and N-His₆-C37 (lanes 2 and 3), N-His₆-C53 (lane 2), N-His₆-FLAG₃-C53 (lane 3), or 2.5 μm N-His₆-TEV-C11(lane 5). After a 10-min incubation, samples were UV-irradiated and processed for SDS-PAGE on 8% (lanes 1–3) or 13% (lanes 4–5) gels as described in under “Experimental Procedures.” C160, C128, C53, and C11 cross-linked products are identified at the right side. The weaker unidentified labeled bands are not pol III subunits. B, C53 cross-links to the nascent transcript of the pol III elongation complex. ABUMP-arrested pol IIIΔ elongation complexes were formed as in A. N-His₆-tagged C53 and C37 were added to 60 nm each for 10 min followed by UV irradiation and Sephacryl S300 chromatography; eluting fractions 3–12 are shown. Cross-linked C160, C128, and C53 are identified at the left side. C, the phosphor image intensity of cross-linked C160 + C128 (□) and C53 (●) is shown normalized to the peak fraction set to 1.0. The undigested nascent transcript also perfectly co-eluted with C160 and C128 (data not shown). A separate mock reaction containing 50 pmol of N-His₆-tagged C53 (—), C37 (not shown), and ³²P-labeled DNA (····) was chromatographed under identical conditions and quantified by Coomassie staining and scintillation counting, respectively, with the peak fraction set to 1.0.

FIGURE 3.

C53 and C11 bind non-specifically to ABUMP-containing RNA. Pol IIIΔ elongation complexes were formed with the 3′-overhang template shown in supplemental Fig. S4 and purified on Sephacryl S300 as specified for Fig. 2. The contained ABUMP-tagged ³²P-labeled RNA (together with unlabeled DNA template) was isolated and aliquots were distributed into buffer CB+100. Non-transcribed DNA template strand or yeast tRNA were added before (for concurrent competition) or 10 min after (for post-competition) the addition of N-His₆-tagged C53 and C37 to 50 nm (upper panel) or N-His₆-TEV-C11 to 2.5 μm (lower panel), as indicated in the figure. Incubation at 20 °C continued for 10 min before UV irradiation and sample processing. The cross-linking efficiency of C53 and C11 relative to the no-competitor control (set to 100) is quantified below each panel. C53, C11, and a protein of size comparable with C37 are identified at the left side. ssDNA, single-stranded DNA.

FIGURE 4.

C53 cross-links to the transcribed DNA strand of the pol IIIΔ elongation complex at register −1/−2. Elongation complexes (+ATP) or mock complexes (no ATP) were formed with the −1/−2 photoactive (ABdUMP) and radioactive DNA probe as described under “Experimental Procedures.” Poly(dI-dC):poly(dI-dC) (to 40 ng/μl) and NaCl (to 0.5 m) were added before chromatography on Sephacryl S300 equilibrated with buffer CB+100 supplemented with 7 mm MgCl₂, with an upper zone of the same buffer containing 0.5 m NaCl. 30-μl volume fractions were taken; to each, poly(dI-dC):poly(dI-dC) was added to 20 ng/μl followed by 30 nm N-His₆-C53, 30 nm N-His₆-C37, and 30 nm N-His₆-TEV-C11. After incubation for 10 min, samples were UV-irradiated and prepared for analysis, as described under “Experimental Procedures.” DNA-cross-linked C160, C128, and C53 are identified at the left side. The asterisks at the right identify cross-linked proteins that are not pol III subunits.

FIGURE 5.

C53 is located at or near the RNA 3′ end of the assembled elongation complex. A, shown are the DNA and RNA components (RNA is italicized) of the assembled elongation complex. B, elongation complexes were assembled with pol IIIΔ with (lanes 5–9) or without (lanes 1–4) added C53 and C37, as described under “Experimental Procedures.” Quantities of 10-mer RNA, transcribed (T), and non-transcribed (NT) DNA strands are specified (in pmol) above each lane. RNA was extended with [α-³²P]CTP (to C13, lane 9) or with [α-³²P]CTP, UTP, and 3′-OMeGTP (to G16, lanes 1–8); the relative yields of these G16 and C13 products are specified below each lane (the line between lanes 4 and 5 designates removal of intervening lanes). C, elongation complexes were assembled and extended to G16, as above, C53 and C37 was added, and samples were UV-irradiated and chromatographed on Sephacryl S300 as described under “Experimental Procedures.” Eluting 60-μl-volume fractions 3–7 comprise the excluded and more included parts of the elution profile. RNA-cross-linked C160, C128, and C53 are identified at the left side. The asterisk at the right identifies a cross-linked protein that is not a pol III subunit.

FIGURE 6.

A role for C53, C37, and C11 in transcriptional initiation; evidence from KMnO₄ footprinting of promoter and elongation complexes. TFIIIB + TFIIIC complexes, initiation complexes, and elongation complexes were assembled on a DNA fragment extending from bp −78 to +161 of the SUP4 tRNA gene. This material was reacted with 20 mm KMnO₄ for 30 s, and samples were processed for analysis of reactive T residues in the SUP4-non-transcribed strand. Values of T-reactivity (cleavage) specified below each lane have been normalized for sample recovery by matching background signal intensity in a nominally non-reactive (duplex) DNA segment (N, at the right of the image). The presence of wild type (wt) pol III, pol IIIΔ, or pol IIIΔ with C53, C37 (25 nm each), and C11 (50 nm) is indicated above each lane. Reactive T residues are identified at the sides.

FIGURE 7.

Pol IIIΔ places additional demands on TFIIIB for initiation of transcription. A, the TATA box of the SNR6 (U6 small nuclear RNA)-derived plasmid pLY1855 permits ambidirectional binding of TFIIIB and generates divergent transcription (see supplemental Fig. S7). Transcription of supercoiled plasmid DNA directed by TFIIIB assembled with wild type (wt, reference type) and mutant Brf1 and Bdp1 is shown. The Brf1 mutation deleting its N-terminal zinc binding domain (residues 1–68; NΔ) was introduced into the background of Brf1(Δ366–408) (rt); removing the latter non-conserved 43-residue segment increases the activity of Brf1 in vitro. The Bdp1 mutation removing residues 355–372 (intΔ) was introduced into the wild type background. TFIIIB-promoter complexes were formed before the addition of wild type pol III or pol IIIΔ, with or without 25 nm C53, C37, and C11 followed in turn by the addition of NTPs for 30 min of multiple-round transcription. Leftward (U6L) and rightward (U6R) transcripts are identified at the left side. Asterisks between lanes 6 and 7 point to the increased length of pol IIIΔ transcripts reflecting defective termination that is corrected by C53 and C37 (lanes 8 and 9). TFIIIB assembled with NΔ68Brf1 is essentially inactive for transcription by pol IIIΔ (lane 7). B, shown is the quantitative presentation of panel A (with U6L and U6R transcripts in lanes 2 and 3 separately normalized to their counterparts in lane 1 and transcripts in lanes 6–9 separately normalized to lane 5) showing partial restoration of activity to pol IIIΔ by C53 and C37, further improved by C11 (less than half of the sample analyzed in panel A, lane 1, was recovered, but this has been corrected relative to its recovery marker in panel B).