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

Alic, Nazif; Ayoub, N.; Landrieux, Emilie; Favry, Emmanuel; Baudouin‐Cornu, Peggy; Riva, Michel; Carles, Christophe

doi:10.1073/pnas.0704116104

Cited by 50 publications

(40 citation statements)

References 47 publications

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“…5B, lane 1). Shorter products appeared to result from misincorporation of UTP in place of CTP, as they did not appear when UTP was absent (forming the 13-mer instead, lane 9) or when the 16-mer was formed by the sequential addition of CTP and UTP (data not shown; note that the addition to misincorporated nucleotides is extremely slow (27)). Curiously, while optimizing the conditions for forming the 16-mer RNA-containing elongation complex (by varying the proportions of the two DNA strands and RNA; compare lane 1 with lanes 2-4), we observed that formation of the 16-mer-RNA elongation complex required the assembly of the non-transcribed DNA strand (although some extension to nt 12 did occur in its absence; data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…1B, lane 1. That their size heterogeneity resulted from slippage was confirmed by walking the entire cluster of bands upon the addition of UTP (lane 2) or UTPϩATP (lane 3; note that most of the complexes in lane 3 extended to C27 instead of A26 due to misincorporation of UMP and the lack of proofreading by pol III⌬ (27) and that ϳ20% of complexes did not chase).…”

Section: Resultsmentioning

confidence: 99%

“…We found that 4-S-UTP was inefficiently incorporated by wild type pol III; weak 4-S-UMP-dependent cross-linking to the C160 and C128 subunits of pol III and to a protein approximately the size of the C53 subunit (ϳ50 kDa) was detected (data not shown). We reasoned that poor incorporation of 4-S-UMP was due to C11-mediated proofreading (27) responding to the anomalous base pairing of 4-S-U. Loss of cross-linking had also been observed when both TFIIS and Mg 2ϩ were added to pol II elongation complexes containing 4-S-U near the nascent RNA 3Ј end (26).…”

Section: Resultsmentioning

confidence: 99%

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The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

Kassavetis

Prakash²,

Shim

2010

Journal of Biological Chemistry

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The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3 end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF. RNA polymerase III (pol III)3 transcribes genes encoding short RNA products that are essential for protein synthesis (e.g. tRNAs, 5 S rRNA), RNA processing (e.g. U6 small nuclear RNA, the RNA subunit of RNase P), protein transport (7SL RNA of the signal recognition particle), and diverse other functions, particularly in higher eukaryotes (1). Pol III is brought to the transcriptional start sites of its genes through interactions with its central 3-subunit initiation factor, TFIIIB (subunits Brf1, TBP, and Bdp1). TFIIIB, in turn, is assembled on DNA upstream of the transcriptional start site in a largely sequenceindependent process by its six-subunit assembly factor, TFIIIC, which binds to two promoter elements (boxA and boxB) located downstream of the transcriptional start site. An additional initiation factor, TFIIIA, serves solely as the adaptor for assembling TFIIIC on 5 S rRNA genes. Vertebrates contain an additional form of TFIIIB in which its paralogue Brf2 replaces Brf1 for transcription of a class of genes that use the SNAP c complex in place of TFIIIC as their TFIIIB-assembly factor (2, 3). Once it has been assembled onto DNA, Saccharomyces cerevisiae TFIIIB suffices for recruiting pol III for robust and accurately initiating transcription in vitro (4); at least in yeast, this also appears to be the case in vivo (5). The complexity of pol III contrasts with the apparent simplicity of its core transcription apparatus (the TFIIIB⅐DNA complex and diverse assembly factors). Five pol III subunits have no counterparts in the considerably less complex pol II (with its merely 12 subunits). Three of these pol III subunits (C82, C34, and C31) form a subassembly that interacts with the TFIIIB⅐DNA complex and is required for specifically initiating transcription (6). Two subunits, C53 and C37, form a subassembly that limits the processivity of pol III elongation and contributes to the abil...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

Kassavetis

Prakash²,

Shim

2010

Journal of Biological Chemistry

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“…One could be initially surprised to find TFIIS associated with class III genes, since Pol III has an intrinsic transcript cleavage activity that depends on Rpc11 subunit (Chedin et al 1998;Alic et al 2007). Rpc11 has a C-terminal Zn loop that bears an RSADE motif, critical for transcript cleavage, that closely resembles the C-terminal domain of TFIIS (Chedin et al 1998).…”

Section: Discussionmentioning

confidence: 99%

Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription

Ghavi-Helm¹,

Michaut²,

Acker³

et al. 2008

Genes Dev.

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TFIIS is a transcription elongation factor that stimulates transcript cleavage activity of arrested RNA polymerase II (Pol II). Recent studies revealed that TFIIS has also a role in Pol II transcription initiation. To improve our understanding of TFIIS function in vivo, we performed genome-wide location analysis of this factor. Under normal growth conditions, TFIIS was detected on Pol II-transcribed genes, and TFIIS occupancy was well correlated with that of Pol II, indicating that TFIIS recruitment is not restricted to NTP-depleted cells. Unexpectedly, TFIIS was also detected on almost all Pol III-transcribed genes. TFIIS and Pol III occupancies correlated well genome-wide on this novel class of targets. In vivo, some dst1 mutants were partly defective in tRNA synthesis and showed a reduced Pol III occupancy at the restrictive temperature. In vitro transcription assays suggested that TFIIS may affect Pol III start site selection. These data provide strong in vivo and in vitro evidence in favor of a role of TFIIS as a general Pol III transcription factor. Gene transcription is a complex and highly regulated process that, in eukaryotes, is carried out by three specialized RNA polymerases (Pol I, II, and III), dedicated to the transcription of different sets of genes. Transcription starts by the assembly of large preinitiation complexes (PIC) comprising TBP (the TATA-binding protein common to all three transcription systems) and general transcription factors specific to the RNA polymerase considered. Other transcription factors facilitate RNA elongation by Pol II (Shilatifard et al. 2003). One of these elongation factors, TFIIS, was initially discovered in human (Natori et al. 1973) and yeast cells (Sawadogo et al. 1980b), and is highly conserved in the eukaryotic and archaeal kingdoms (Hausner et al. 2000;Fish and Kane 2002). Structurally unrelated but functionally equivalent factors (GreA and GreB) also operate in bacteria (Borukhov et al. 1993;Opalka et al. 2003). TFIIS forms a binary complex with Pol II and stimulates an intrinsic transcript cleavage activity of RNA polymerase allowing elongating enzymes to resume RNA synthesis after accidental transcription arrest (Fish and Kane 2002;Kettenberger et al. 2003).TFIIS is organized in three domains as determined by limited proteolysis and nuclear magnetic resonance (NMR) (Awrey et al. 1997;Olmsted et al. 1998). The three-dimensional structure of the Pol II-TFIIS complex, without the TFIIS N-terminal domain, has been determined by crystallographic studies (Kettenberger et al. 2003). The Nterminal domain I of TFIIS protrudes from Pol II and is not required for elongation (Nakanishi et al. 1995;Awrey et al. 1998), but binds the yeast Mediator and SAGA coactivator (Wery et al. 2004). The TFIIS central domain II is inserted into the funnel-shaped pore of Pol II and brings the C-terminal conserved RSADE motif very close to the enzyme active site (Kettenberger et al. 2003). Remarkably, the C-terminal domain III of TFIIS shares the RSADE motif with the C-terminal parts o...

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“…Indeed, repetition of our cleavage assay with a scaffold that contains only a single mismatch at the RNA 3 0 end, mimicking the situation after a misincorporation event, induced efficient RNA cleavage (not shown). For Pol III, the intrinsic cleavage activity was recently shown to enable proofreading in a manner dependent on the A12.2 homolog C11 (Alic et al, 2007), which is required for the intrinsic cleavage activity of Pol III (Chedin et al, 1998;Landrieux et al, 2006).…”

Section: Possible Functions Of the Cleavage Activitymentioning

confidence: 99%

Functional architecture of RNA polymerase I

Geiger¹,

Kuhn²,

Baumli³

et al. 2009

Acta Cryst Sect A

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SUMMARYSynthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 Å cryoelectron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3 0 -RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3 0 -end trimming.

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

Cited by 50 publications

References 47 publications

The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription

Functional architecture of RNA polymerase I

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