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Mechanism of Eukaryotic RNA Polymerase III Transcription Termination

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Science  28 Jun 2013:
Vol. 340, Issue 6140, pp. 1577-1580
DOI: 10.1126/science.1237934

Stopping Transcription

It is as important to terminate any biological process as it is to start it. Transcription, copying information encoded in genes into RNA, requires accurate and timely termination. Nielsen et al. (p. 1577) present a mechanism for transcription termination by RNA polymerase III, the enzyme that synthesizes the majority of RNA molecules in eukaryotes. In this scenario, the folding of the RNA as it is transcribed by polymerase into a highly structured transcript causes termination at the end of its synthesis. This mechanism may serve as a control of proper folding of structural or catalytic RNAs synthesized by RNA polymerase III. Comparison with other organisms suggests that this mechanism emerged before divergence of bacteria and eukaryotes.

Abstract

Gene expression in organisms involves many factors and is tightly controlled. Although much is known about the initial phase of transcription by RNA polymerase III (Pol III), the enzyme that synthesizes the majority of RNA molecules in eukaryotic cells, termination is poorly understood. Here, we show that the extensive structure of Pol III–synthesized transcripts dictates the release of elongation complexes at the end of genes. The poly-T termination signal, which does not cause termination in itself, causes catalytic inactivation and backtracking of Pol III, thus committing the enzyme to termination and transporting it to the nearest RNA secondary structure, which facilitates Pol III release. Similarity between termination mechanisms of Pol III and bacterial RNA polymerase suggests that hairpin-dependent termination may date back to the common ancestor of multisubunit RNA polymerases.

Termination of transcription is an obligatory step after synthesis of the transcript, which leads to dissociation of RNA polymerase (RNAP) and the transcript from the template DNA. However, evolutionarily conserved multisubunit RNAPs from bacteria and Archaea and three eukaryotic RNAPs use different mechanisms to terminate transcription (13). Eukaryotic polymerase III (Pol III) terminates after synthesis of a poly-U stretch (4, 5), and most studies have focused on the efficiency of recognition of the poly-T (on the nontemplate strand) termination signal (6, 7). However, the events leading to termination on the poly-T signal; that is, dissociation of Pol III from the template, are not known.

We used in vitro–assembled elongation complexes, which have been successfully used to investigate various RNAPs (811), to examine this problem. These complexes—assembled with purified RNAP, synthetic complementary template and nontemplate DNA strands, and RNA—allow us to skip the initiation step, therefore excluding any accessory factors from the reaction. We immobilized complexes on streptavidin beads through biotin on the 5′ end of the nontemplate strand, and we labeled RNA by incorporation of radioactive nucleoside monophosphates (scheme in Fig. 1A) (12). We analyzed transcription through poly-T signals of various lengths by purified Saccharomyces cerevisiae Pol III. As seen in Fig. 1, A and B, transcripts finishing after T6 to T10 were transcribed (compare to fig. S1).

Fig. 1 Pol III pauses on the poly-T signal but does not terminate.

(A) Scheme of assembled elongation complexes (EC13) containing 13-nt-long RNA (RNA13) is shown at the top. RNA was radiolabeled at the 3′ end of guanosine monophosphates (bold) (12). Complexes were immobilized on beads through biotin on the 5′ end of the nontemplate strand. Transcription occurred for 10 min on templates with poly-T signals of different lengths in the presence of 1 mM of either uridine triphosphate (UTP) or all nucleoside triphosphates (NTPs). Hereafter, black vertical lines separate parts of a single gel that were brought together. (B) After 10 min of transcription on the templates depicted above the gels (12), released transcripts (supernatant, “super”) were separated from transcripts that remained in the immobilized complexes (“beads”) (scheme in the frame above the gels). The length of RNA preceding the poly-U tract is depicted above the gels.

To test if transcripts ending with a poly-U stretch were released from the template as a result of termination, we analyzed RNA in the supernatant and immobilized fractions of the reaction (scheme of Fig. 1B). As seen in Fig. 1B, although RNAs resulting from transcription to the end of template (run-off products) were released in the supernatant, transcripts ending at the poly-T signal remained part of the elongation complex (even after prolonged incubation), independently of the length of the transcript or sequences surrounding the poly-T signal (12). The inability of Pol III and RNA to leave DNA was not due to the deficiency in the upstream DNA duplex restoration or to formation of an extended RNA-DNA hybrid (13) (fig. S2, A and B). These results indicate that Pol III pauses rather than terminates at the poly-T signal.

We analyzed transcription of the full-length 5S and tRNATyr (SUP4) genes. Given the length of each gene, as a template we used streptavidin-bead–immobilized, double-stranded polymerase chain reaction product with a single-stranded extrusion at the 3′ end of the template strand and an RNA primer complementary to this extrusion (12, 14) (scheme in Fig. 2A). Transcription ended in the poly-T signal; however, the full-length 5S and tRNATyr RNAs were readily released from the template (Fig. 2A, lanes 1 to 3 and 7 to 9, respectively). The abolished release of the run-off products is explained by immobilization of complexes via the 5′ end of the template strand.

Fig. 2 Termination by Pol III is facilitated by the secondary structure of the transcript.

(A and B) Termination by Pol III on full-length genes and their mutant variants lacking secondary structure before poly-U of the transcripts [lacking hairpin (-HP) 5S-HP and tRNATyr-HP or having unstructured spacer (+UN) tRNATyr+UN] (12). Here and below, release was analyzed after 1 min. (A) Transcription was initiated by purified Pol III on the construct with the single-stranded overhang (scheme above the gels). dsDNA, double-stranded DNA. (B) Transcription was performed in yeast nuclear lysate on templates carrying the promoter (scheme shown above the gels; TATA, Box A, and Box B are Pol III promoter elements bound by transcription factors, TF). (C) Absence of release of transcript without a hairpin (lanes 1 to 3) and release of transcripts containing an arbitrary hairpin (lanes 4 to 6), an RNA duplex formed by an externally added oligonucleotide (lanes 7 to 9), or helix I of 5S RNA [lanes 10 to 12; see also the scheme in (A)] before the poly-U tract (12). bp, base pair. (D) Termination of transcripts bearing spacers of different lengths between the poly-U tract and the termination hairpin (5S helix I) (12). See also fig. S4.

Transcript release during bacterial transcription termination is facilitated by an RNA hairpin that forms behind the poly-U tract (15). All transcripts synthesized by Pol III, as per their functions (structural or tRNAs), have extensive secondary structures, so that the poly-U tract is preceded by RNA hairpins and/or stems (fig. S3 and table S1). We therefore hypothesized that, as in the case of bacterial termination, termination by Pol III may also be facilitated by an RNA hairpin and/or stem, in this case, provided by the structure of RNA itself. To test this hypothesis, we changed the sequence of the 5S and tRNATyr genes to eliminate formation of RNA secondary structures close to poly-U stretch of the transcript (5S-HP and tRNATyr-HP templates; -HP, no hairpin). The release of the transcripts ending in the poly-U signals of the mutant genes was indeed abolished (Fig. 2A, lanes 4 to 6 and 10 to 12).

To test the requirement for the RNA secondary structure during Pol III termination in the presence of transcription factors, we analyzed transcription in more native conditions by using S. cerevisiae nuclear lysates and promoter-containing DNA templates (scheme in Fig. 2B). Given that any alterations to the secondary structure of tRNATyr destroy the internal promoter of Pol III, an unstructured spacer was introduced between the body of tRNATyr and the poly-U stretch. In full agreement with the results obtained with purified Pol III, the secondary structure preceding the poly-U tract was essential for termination (Fig. 2B).

To directly test the role of an RNA hairpin in termination by Pol III, we changed the sequence of template T12, which did not allow for release (Fig. 2C, lanes 1 to 3), so that the synthesized transcript formed a 9–base pair–long hairpin before the poly-U stretch. This led to the release of the transcript ending in the poly-T signal; that is, to termination (HP/T12 template; Fig. 2C, lanes 4 to 6). Addition of a short RNA complementary to the hairpin-less transcripts upstream of (but not far away from; see fig. S4) the poly-U stretch, which mimics a termination hairpin (16), also resulted in efficient termination (Fig. 2C, lanes 7 to 9). The 5S helix I, formed by the most proximal 5′ and 3′ parts of 5S RNA (scheme in Fig. 2A), placed upstream of the poly-T signal also caused efficient termination (Fig. 2C, lanes 10 to 12).

For efficient termination by bacterial RNAP, an RNA hairpin has to be immediately upstream of the poly-U stretch (1, 17). Consistently, a hairpin immediately upstream in the poly-U tract also causes termination by Pol III (Fig. 2C). To test the requirements for the distance between a hairpin and the poly-U tract, we introduced unstructured spacers of different lengths (Fig. 2D). We found that a distance as large as ~12 base pairs between the poly-U stretch and the hairpin allows for efficient termination (Fig. 2D, lanes 1 to 3). Longer spacers result in diminished termination (Fig. 2D, lanes 4 to 9; see also fig. S4). These results suggest that an RNA hairpin formed within ~12 nucleotides (nt) upstream of the poly-U stretch is sufficient for termination of the poly-T–paused complex, which is consistent with lengths of spacers between the poly-U tract and the nearest secondary structure found in transcripts synthesized by Pol III (table S1).

The ability of the termination hairpin to act on the paused complex at a distance of ~20 nt from the 3′ end of the transcript suggests that the paused complex should slide backward to approach the hairpin. We analyzed the geometry of the paused elongation complexes carrying 8U (EC8U) and 10U (EC10U) tracts on the 3′ ends of their hairpin-less transcripts. To map the position of the Pol III active center, we used the ability of the RNAP active center to immobilize Fe2+ ion (instead of the native Mg2+), which, by generating hydroxyl radicals, induces cleavage of the transcript in the vicinity of the active center (18). As seen in Fig. 3A, transcripts in EC8U and EC10U were cleaved in the 5′ proximal part of the poly-U tract (lanes 4 and 9), indicating that the active center of Pol III has backtracked from the 3′ end of RNA. Protection from ribonuclease A (RNase A), which cleaves single-stranded RNA after pyrimidines, was also consistent with the backtracked conformation, as bodies and poly-U tracts of the transcripts of EC8U and EC10U were mostly protected from RNase A (Fig. 3A, lanes 2 and 7, and fig. S5). In agreement with the length of the secondary channel (19), the 3′ end proximal Us in EC10U, but not in EC8U, were exposed to RNase A (Fig. 3A, lanes 2 and 7, and fig. S5). Backtracking of the termination complex thus explains a loose (anywhere within ~12 nt) requirement for the positioning of the termination hairpin and/or stem upstream of the poly-T signal.

Fig. 3 Complex paused on termination signal undergoes deep backtracking.

(A) Probing of EC8U and EC10U with RNase A and hydroxyl radicals generated by Fe2+ bound in the Pol III active center (scheme below the gels). Lanes 5 and 10 (without dithiothreitol, DTT) are controls for hydrolysis caused by Fe2+. Radiolabels in transcripts are shown in red. Cleaved positions are shown with arrows. The identity of positions cleaved by RNase A was confirmed with 5′-end–labeled RNA (fig. S5). Interpretation of the probing results is shown schematically below the gels. (B) RNA extension and hydrolysis in EC8U, EC10U, and EC8A (see also fig. S6).

Note that the backtracking of ECpolyU is unusual because the highly efficient hydrolytic activity of the Pol III active center (14), which can rescue a backtracked complex, is switched off (compare complexes in Fig. 3B and fig. S6B). Such unusual inactivation, as well as impossibility of RNA extension in ECpolyU (compare complexes in Fig. 3B and fig. S6B), ensures the formation of a “dead-end” complex, whose only fate is to terminate.

The distance between the Pol III active center (but not necessarily the 3′ end of RNA) and the RNA secondary structure required for termination is 7 nt (fig. S7A and supplementary text), which, notably, resembles bacterial termination. Most of the Pol III transcripts contain no or very short spacers between the poly-U signal and the nearest secondary structure (table S1). Therefore, the function of poly-T signal on these templates is to pause Pol III at ~7 nt from the nearest secondary structure (supplementary text and fig. S8). However, some transcripts synthesized by Pol III contain longer spacers between the poly-U tract and the nearest RNA duplex (fig. S3 and table S1), which suggests that the deep backtracking on the poly-T signal of these genes is required to bring Pol III closer to the nearest secondary structure (fig. S8). In the case of short spacers, the 3′ penultimate RNA duplex (such as helix IV of 5S RNA and the TψC arm of tRNA) can also cause termination, should the 3′ proximal hairpin fail to fold and Pol III backtracks (fig. S8).

Backtracking on the poly-T signal results in a strong (G- and C-rich) RNA-DNA hybrid within the termination complex but does not influence termination. Efficient hairpin-dependent termination can also be achieved on a poly-G track (fig. S7A). Therefore, termination by Pol III does not require a weak RNA-DNA hybrid, as was postulated earlier (15). Backtracking and the presence of a nonhomopolymeric RNA-DNA hybrid within the backtracked ECpolyU also exclude possibilities of forward translocation (20) and RNA-DNA hybrid shearing (21), respectively, as possible mechanisms for termination. The results are consistent with the recently proposed allosteric mechanism of termination (22), when the RNA hairpin allosterically opens RNAP and leads to its dissociation from the template, though we cannot exclude the possibility that hairpin-dependent destruction of the Pol III elongation complex takes place via a different route.

The above results argue that, in itself, the poly-T signal may not be sufficient for elongation complex destruction. Archaeal RNAP, which was proposed to terminate on the poly-T signal without involvement of additional factors (3), also fails to dissociate on the poly-T signal, whereas an RNA hairpin is sufficient to cause termination (fig. S7B). This finding suggests that Archaea may also use RNA-duplex–dependent termination, the mechanism of which may date back to the last universal common ancestor (LUCA). Termination caused by structures embedded in the functional body of the transcript provides a simple, factor-independent mechanism for the finish of gene transcription and may serve as a checkpoint for proper folding of RNA, which has been essential for the ribozymes of the LUCA and remains essential for structural and/or catalytic RNAs synthesized by Pol III.

Supplementary Materials

www.sciencemag.org/cgi/content/full/340/6140/1577/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S8

Table S1

References (2327)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: This work is dedicated to the memory of Denis Izyumov. We thank J. Roberts, E. Nudler, P. E. Geiduschek, and J. Brown for critical reading of the paper; G. Kassavetis, R. van Nues, H. Murray, N. Proudfoot, and P. Braglia for experimental advice; and J. Chong for help with Archaea. This work was supported by the UK Biotechnology and Biological Sciences Research Council, the Biotechnology and Biological Sciences Research Council under the SysMO initiative, and the European Research Council [grant ERC-2007-StG 202994-MTP]. Data are available in the supplementary materials.
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