A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation

Abstract

While initiation of transcription by RNA polymerase III (Pol III) has been thoroughly investigated, molecular mechanisms driving transcription termination remain poorly understood. Here we describe how the characterization of the in vitro transcriptional properties of a Pol III variant (Pol IIIdelta), lacking the C11, C37, and C53 subunits, revealed crucial information about the mechanisms of Pol III termination and reinitiation. The specific requirement for the C37-C53 complex in terminator recognition was determined. This complex was demonstrated to slow down elongation by the enzyme, adding to the evidence implicating the elongation rate as a critical determinant of correct terminator recognition. In addition, the presence of the C37-C53 complex required the simultaneous addition of C11 to Pol IIIdelta for the enzyme to reinitiate after the first round of transcription, thus uncovering a role for polymerase subunits in the facilitated recycling process. Interestingly, we demonstrated that the role of C11 in recycling was independent of its role in RNA cleavage. The data presented allowed us to propose a model of Pol III termination and its links to reinitiation.

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription

“…1B, lanes 3-5). In contrast to this apparent inability of Pol III to incorporate a mispaired nucleotide, Chédin et al (25) have reported that transcription of SUP4 in the absence of GTP by Pol III⌬, an incomplete form of Pol III lacking the C11, C37 and C53 subunits, resulted in formation of an 18-mer transcript differing at the 3Ј end from the 17-mer produced by Pol III (25,26). The authors suggested that Pol III⌬ incorporated a mispaired nucleotide at position ϩ18, which failed to be subsequently removed because of the lack of RNAcleavage activity in Pol III⌬ resulting from the absence of the TFIIS-like C11 subunit (25).…”

“…The SUP4 gene was transcribed in the absence of GTP by Pol III⌬ preincubated with the recombinant C11 subunit (rC11). The resulting halted ternary complexes contained a 17-mer RNA and were purified to remove rC11 and the NTPs (25,26). Traces of the 18-mer transcript remained, which could be attributed to a somewhat reduced cleavage efficiency of rC11 compared with the endogenous C11, and a negligible amount of a slippage product (27) was observed (Fig.…”

Transcription termination by nuclear RNA polymerases

“…In vitro transcription using RNAPIII lacking the RNA-PIII-specific subunits C11, C37, and C53 (RNAPIIID) can support transcription initiation but fails to terminate properly (Landrieux et al 2006). Adding back the heterodimer C37/C53 was sufficient to allow recognition of the terminator and to correct the termination defect, but not for transcription reinitiation.…”

“…Landrieux et al (2006) showed that C11 RNA cleavage activity is not required for correct recognition of the RNAPIII terminator elements and release of the newly synthesized transcript. However, C11 itself, but not its cleavage activity, is required for reinitiation (Chedin et al 1998;Landrieux et al 2006). These experiments strongly suggest that termination and cleavage of the RNA precursor are not coupled in the RNAPIII transcription process.…”

“…C11 is an essential subunit that mediates the intrinsic RNA cleavage activity of RNAPIII (Whitehall et al 1994) and shows homology with the RNAPII subunit Rpb9 and to RNAPII elongation factor TFIIS (Chedin et al 1998). Landrieux et al (2006) showed that C11 RNA cleavage activity is not required for correct recognition of the RNAPIII terminator elements and release of the newly synthesized transcript. However, C11 itself, but not its cleavage activity, is required for reinitiation (Chedin et al 1998;Landrieux et al 2006).…”