Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

Abstract

Eukaryotic RNA polymerase (Pol) III terminates transcription at short runs of T residues in the coding DNA strand. By genomic analysis, we found that T 5 and T 4 are the shortest Pol III termination signals in yeasts and mammals, respectively, and that, at variance with yeast, oligo(dT) terminators longer than T 5 are very rare in mammals. In Saccharomyces cerevisiae, the strength of T 5 as a terminator was found to be largely influenced by both the upstream and the downstream sequence context. In particular, the CT sequence, which is naturally present downstream of T 5 in the 3-flank of some tDNAs, was found to act as a terminator-weakening element that facilitates translocation by reducing Pol III pausing at T 5 . In contrast, tDNA transcription termination was highly efficient when T 5 was followed by an A or G residue. Surprisingly, however, when a terminationproficient T 5 signal was taken out from the tDNA context and placed downstream of a fragment of the SCR1 gene, its termination activity was compromised, both in vitro and in vivo. Even the T 6 sequence, acting as a strong terminator in tRNA gene contexts, was unexpectedly weak within the SNR52 transcription unit, where it naturally occurs. The observed sequence context effects reflect intrinsic recognition properties of Pol III, because they were still observed in a simplified in vitro transcription system only consisting of purified RNA polymerase and template DNA. Our findings strengthen the notion that termination signal recognition by Pol III is influenced in a complex way by the region surrounding the T cluster and suggest that read-through transcription beyond T clusters might play a significant role in the biogenesis of class III gene products.Eukaryotic RNA polymerase (Pol) 1 III is unique among DNAdependent RNA polymerases in recognizing a simple run of T residues on the coding strand as a termination signal, in the apparent absence of accessory factors. The minimal signal thought to be sufficient to provoke Pol III termination varies among different eukaryotes. T 4 suffices for termination by Xenopus and human Pol III (1, 2), T 4 /T 5 for Schizosaccharomyces pombe Pol III (3), and T 5 /T 6 for Saccharomyces cerevisiae Pol III (4). Pol III termination within T clusters is generally heterogeneous and progressive (5), and termination efficiency tends to increase with the length of the T run (4). Mechanistically, Pol III termination involves extensive pausing, dictated by the T cluster itself (5, 6). Even during elongation, the addition of three UMP residues in succession is particularly slow (5). Pol III pausing at T clusters sets cycles of hydrolytic RNA chain retraction (7). Both pausing and the associated hydrolytic RNA cleavage are affected by mutations in the C160, C128, and C11 subunits of yeast Pol III (7-9). In the case of C128 and C11, the same mutations have also been shown to affect the termination properties of Pol III, thus suggesting a functional interplay between hydrolytic RNA cleavage and transcription termination (9, 10). The t...

Transcription termination by nuclear RNA polymerases

“…RNAPIII recycling is also dependent on a natural termination signal (Dieci and Sentenac 1996;Ferrari et al 2004). Termination can be influenced by sequences surrounding the T tract (Bogenhagen and Brown 1981;Mazabraud et al 1987;Gunnery et al 1999;Braglia et al 2005). Braglia et al (2005) proposed that the surrounding residues and, in particular, downstream residues of the yeast terminator influence the pausing time or the processivity of RNA-PIII.…”

“…In yeast, the most frequent T stretch length is six or seven with no stretch shorter than five Ts (Allison and Hall 1985;Hamada et al 2000). In mammals, a stretch of four Ts is the most frequent terminator, and a stretch of more than five Ts is rare (Braglia et al 2005). RNAPIII pauses not only at the terminator region but also at several other T stretches during transcription (Campbell and Setzer 1992;Matsuzaki et al 1994).…”

“…Termination can be influenced by sequences surrounding the T tract (Bogenhagen and Brown 1981;Mazabraud et al 1987;Gunnery et al 1999;Braglia et al 2005). Braglia et al (2005) proposed that the surrounding residues and, in particular, downstream residues of the yeast terminator influence the pausing time or the processivity of RNA-PIII. However, no consistent pattern for this context effect has been elucidated.…”

A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III

“…A curious feature of the transcription unit is the presence of a run of 6 consecutive T residues located a few bp downstream of the box A. Even though the T 6 sequence is a strong termination signal for Pol III, we have recently shown that it behaves as an unusually weak terminator in the SNR52 context (33). The T 6 element might be relevant for SNR52 expression, because it is also present in this position in the genomes of at least two other Saccharomyces species (S. paradoxus and S. mikatae).…”

“…In Vitro Transcription Assays-Transcription of class III genes was reconstituted in vitro essentially as described (15,33). All reactions contained 0.6 g of TFIIIC partially purified up to the DEAE Sephadex A-25 step (34), except that yeast nuclear extract (35) was used as a starting material; 40 ng of recombinant TBP and 80 ng of recombinant Brf1, both purified from overexpressing Escherichia coli cells (34); and 10 ng of highly purified RNA polymerase III (15).…”