Mechanism of eukaryotic RNA polymerase III transcription termination

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.

Fig. 1

Pol III pauses on the poly-T signal but does not terminate. A. Scheme of assembled elongation complexes is shown at the top. RNA was radiolabelled at the 3′ end GMPs (bold) (12). Complexes were immobilized on beads via biotin on the 5′ end of the non-template strand. Transcription for 10 min on templates with poly-T signals of different lengths in the presence of 1 mM either UTP or all NTPs. Here and after black lines separate parts of one gel that were brought together. B. After 10 min transcription on the templates depicted above gels (12), released transcripts (“super”) were separated from transcripts that remained in the immobilized complexes (“beads”) (scheme in the frame above the gels). Length of RNA preceding poly-U tract is depicted above the gels.

Fig. 2

Termination by Pol III is facilitated by the secondary structure of the transcript. A, B. Termination by Pol III on full-length genes and their mutant variants lacking secondary structure before poly-U of the transcripts (5S-HP and tRNA^Tyr-HP, tRNA^Tyr+UN) (12). Release was analyzed after 1 min of transcription. A. Transcription was initiated by purified Pol III on the construct with the single-stranded overhang (scheme above the gels). B. Transcription was performed in yeast cell lysate on templates carrying promoter (promoter elements and transcription factors schematically shown above the gels). Secondary structure of tRNA^Tyr transcript could not be altered as in panel A, because it would destroy the internal promoter of Pol III. C. Absence of release of transcript without hairpin (lanes 1-3), and release of transcripts containing arbitrary hairpin (lanes 4-6), duplex formed by externally added RNA oligonucleotide (lanes 7-9) or helix I of 5S RNA (lanes 10-12; see also scheme in panel A) before the poly-U tract (12). Release was analyzed after 1 min of transcription. D. Termination of transcripts (after 1 min of transcription) bearing spacers of different length between poly-U and termination hairpin (5S helix I) (12). See also Fig. S4.

Fig. 3

Complex paused on termination signal undergoes deep backtracking. A. Probing of paused complexes containing 8U and 10 U tracts at the 3′ ends of transcripts 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 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. S6). Interpretation of the probing results is shown schematically below the gels. B. RNA extension and hydrolysis in paused complexes containing 8U,10U and 8A tracks at the 3′ end of RNA (see also Fig. S6). Radiolabels in transcripts are shown in red.