RNA polymerase III subunits C37/53 modulate rU:dA hybrid 3′ end dynamics during transcription termination

Abstract

RNA polymerase (RNAP) III synthesizes tRNAs and other transcripts, and mutations to its subunits cause human disorders. The RNAP III subunit-heterodimer C37/53 functions in initiation, elongation and in termination-associated reinitiation with subunit C11. C37/53 is related to heterodimers associated with RNAPs I and II, and C11 is related to TFIIS and Rpa12.2, the active site RNA 3′ cleavage factors for RNAPs II and I. Critical to termination is stability of the RNA:DNA hybrid bound in the active center, which is loose for RNAP III relative to other RNAPs. Here, we examined RNAP III lacking C37/53/C11 and various reconstituted forms during termination. First, we established a minimal terminator as 5T and 3A on the non-template and template DNA strands, respectively. We demonstrate that C11 stimulates termination, and does so independently of its RNA cleavage activity. We found that C37/53 sensitizes RNAP III termination to RNA:DNA hybrid strength and promotes RNA 3′ end pairing/annealing with the template. The latter counteracts C11-insensitive arrest in the proximal part of the oligo(T)-tract, promoting oligo(rU:dA) extension toward greater hybrid instability and RNA release. The data also indicate that RNA 3′ end engagement with the active site is a determinant of termination. Broader implications are also discussed.

Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription

“…RNAP3 is predominantly implicated in the transcription of the short and abundant tRNA and 5S rRNA species. In vitro transcription assays have indicated that once loaded, RNAP3 can terminate transcription autonomously upon reaching a transcription termination signal, which is constituted by a simple stretch of five thymine residues on the non-template strand (Mishra & Maraia, 2019), although this number may vary for different genes and organisms (reviewed in Arimbasseri et al 2013). TFIIIB in turn recruits the 17 subunits of RNAP3 complex at the TSS to initiate transcription (reviewed in Schramm and Hernandez 2002).…”

“…In fission yeast, an upstream TATA box assists TFIIIC in recruiting TFIIIB and is essential for the proper recruitment of RNAP3 (Hamada et al, 2001). In vitro transcription assays have indicated that once loaded, RNAP3 can terminate transcription autonomously upon reaching a transcription termination signal, which is constituted by a simple stretch of five thymine residues on the non-template strand (Mishra & Maraia, 2019), although this number may vary for different genes and organisms (reviewed in Arimbasseri et al 2013). The C37/53/11 subcomplex of RNAP3 is particularly important for this intrinsic transcription termination mechanism (Arimbasseri et al, 2013).…”

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential

“…Instead, the low GC content of the genomic regions from which these RNA species arise inherently causes RNAPII to be termination prone. Indeed, high AT content increases RNAPII pausing and backtracking 29 , and promotes termination by other RNA polymerases [30][31][32] , since AT-rich RNA-DNA hybrids destabilize the ternary elongation complex. In addition, AT-rich RNAs fail to form stable secondary structures that prevent RNAPII backtracking and arrest 33,34 .…”

Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

“…2b, Supplementary Video 1). Multiple studies reported that Pol III requires RPC10 (C11 in yeast) to terminate transcription, but that its 3' RNA cleavage activity is not essential for this process 9,11,12,27,28 . We propose that the insertion of RPC10 C-ribbon induces partial opening of the mobile clamp domain and, thereby, supports the release of transcribed RNA from a paused pre-termination complex previously reported 10 .…”