The Presence of an RNA:DNA Hybrid That Is Prone to Slippage Promotes Termination by T7 RNA Polymerase

Abstract

Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine (poly(U)) tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions) T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a “locked” complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.

The P1N-PISPO trans -Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor

“…This mechanism of expression is supported by examination of sequences corresponding to different potyviruses present in the virome of the AM-MB2 plant, which showed results consistent with polymerase slippage in equivalent G 2 A 6 motifs for SPV2 and SPVC (see Table S7 in the supplemental material). Moreover, our experiments designed to identify translational frameshifting in WGE failed to show a noticeable production of truncated frameshifted protein products, and the presence of minor products compatible in size could be also generated by T7 polymerase slippage, as shown by others (14,49,50). Consistent with our view that viral RNA polymerase slippage is the most likely mechanism of production of out-of-frame products in potyviruses, the transient expression of wild-type P1 sequence out of the viral infection context resulted in the production only of P1-derived peptides when analyzed with the sensitive mass spectrometry technique (Fig.…”

Mechanism of Transcription Termination by RNA Polymerase III Utilizes a Non-template Strand Sequence-Specific Signal Element

“…S1). The heterogeneity of readthrough (RT) transcripts raised the possibility of transcript slippage within the homopolymeric terminator (Molodtsov et al, 2014; Strathern et al, 2013). However, a template in which the 9T terminator was replaced by a mixed nucleotide sequence showed similar RT heterogeneity (Fig.…”

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators

“…Interestingly, with phage T7 single-subunit RNAP, an RNA stem loop structure (TΦ) stimulates intrinsic termination on the upstream RNA-DNA hybrid containing poly(A) or polyU tracts. In the absence of a stem loop, T7 RNAP slips on the same motifs leading to nt addition(s), but this slippage is strongly inhibited by stem loop formation (50). In vitro, a small fraction of the TEC C4 also terminates transcription.…”