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, while not causing 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 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 multi-subunit RNA polymerases.Termination of transcription is an obligatory step following synthesis of the transcript, which leads to dissociation of RNA polymerase (RNAP) and the transcript from the template DNA. However, apparently different mechanisms are utilized by evolutionary conserved multi-subunit RNAPs from bacteria, archaea, and three eukaryotic RNAPs to terminate transcription (1-3). 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). Both upstream and downstream sequences were shown to influence efficiency of recognition (7). However, the events leading to termination on the poly-T signal, i.e. dissociation of Pol III from the template, are not known.We investigated this problem by using assembled elongation complexes, a technique successfully used to investigate various RNAPs (8-11). These complexes, assembled with purified RNAP, synthetic complementary template and non-template DNA strands and RNA, allow skipping the step of initiation and, therefore, excluding any accessory factors from the reaction. Complexes were immobilized on streptavidin beads via biotin on the 5′ end of the non-template strand (scheme in Fig. 1A). The RNA in complexes was radioactively labeled by incorporation of radioactive NMP (12). First, we analyzed transcription through poly-T signals of various lengths by purified S. cerevisiae Pol III. As seen from Fig. 1A, at poly-T signals longer than 5 nucleotides, transcripts finishing at the end the poly-T signal were formed. On long poly-T signals (12T), transcription was stopping predominantly after 6 th -10 th T (T 12 template in Fig. 1B, lane 10). No stopping was observed on homopolymeric tracts other than poly-T (Fig. S1). We tested, if transcripts ending with a poly-U stretch were released from the template as a result of termination. This can be done by analysis of transcripts in the supernatant and immobilized fractions of the reaction ("super" and "beads" fractions, respectively, in scheme of Fig. 1B). As seen from Fig. 1B, while RNAs resulting from transcription to the end of tem...

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

“…In particular, mammalian RNAP II was shown to respond to the bacterial consensus pause signal (30) and yeast RNAP III was suggested to recognize RNA hairpins during termination (58). Furthermore, an open, ratcheted RNAP conformation was observed in eukaryotic RNAP I (59,60) and in archaeal RNAP (61).…”

RNA polymerase I structure and transcription regulation

“…Further backtracking may occur during termination and enable cleft expansion and hybrid release, as suggested for the bacterial enzyme 45 and for Pol III (ref. 46). By contrast, the tunable active site of Pol II allows for accommodation of backtracked RNA in the pore, Pol-II-specific regulation during the elongation phase, and 39 RNA processing on the polymerase surface by the machinery for pre-mRNA cleavage and polyadenylation.…”

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

“…Overall, analysis of known phage and cellular factors suggests that they apparently use a limited number of strategies to control transcription termination through suppression of the transcription pausing and prevention of the RNA hairpindependent TEC dissociation. Because eukaryotic RNAPs can respond to the hairpin termination signals in vitro (46,47), one can expect that similar termination and antitermination pathways may be discovered in eukaryotic gene expression.…”