A universally conserved region of the largest subunit participates in the active site of RNA polymerase III.

Dieci, Giorgio; Denmat, Sylvie Hermann‐Le; Lukhtanov, Evgeny A.; Thuriaux, Pierre; Werner, Michel; Sentenac, André

doi:10.1002/j.1460-2075.1995.tb00046.x

Cited by 71 publications

(89 citation statements)

References 46 publications

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“…1A) contains a 4-nt 3Ј-overhang at one end for factor-independent transcription (28) initiating with the dinucleotide primer GpA followed in turn by a run of 15 AMP residues, CMP ( 32 P-labeled), and the photoactive UMP analogue. We expected the initial run of weakly base-pairing A residues to result in reiterative slippage during repetitive addition of AMP to the initially synthesized RNA, generating a cluster of transcripts differing in length but with identical 3Ј ends (as previously encountered with E. coli RNA polymerase and also seen to occur with pol III (29,30)). The array of nascent transcripts contained within elongation complexes formed in the absence of UTP is shown in Fig.…”

Section: Resultsmentioning

confidence: 97%

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: 97%

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 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. 1B, lane 6).…”

Section: Resultsmentioning

confidence: 99%

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

Alic

Ayoub²,

Landrieux³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10 3 over the error rate determined solely by selectivity (1.8 ؋ 10 ؊4 ). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.nucleotide selectivity ͉ transcriptional fidelity

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“…S3B). Although these two positions are not directly engaged in the coordination of the Mg 2+ ions that participate in catalysis and can tolerate conservative substitutions (Dieci et al 1995), the structure of the RNAPII elongation complex has recently suggested a possible role for the first residue N in the specificity for ribo-rather than deoxyribonucleotide (Gnatt et al 2001). Whether the amino acid substitutions found at this position in NRPD1a and NRPD1b are indicative of a more relaxed specificity of the RNAPIV enzyme(s) toward the nucleotide substrate or reveal a more specific adaptation to novel function remains to be de- termined.…”

Section: Arabidopsis Expresses Two Class IV Largest Subunit Genesmentioning

confidence: 99%

Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis

Pontier¹,

Yahubyan²,

Véga³

et al. 2005

Genes Dev.

359

441

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Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production. A major evolutionary distinction separating prokaryotes from eukaryotes is the passage from a unique multisubunit DNA-dependent RNA polymerase enzyme (RNAP) to three complexes (Roeder and Rutter 1969), each responsible for the transcription of a subclass of nuclear DNA sequences (Sentenac 1985). RNA polymerase I (RNAPI) transcribes the repeated genes encoding the large ribosomal RNAs, which represent up to four-fifths of total RNA. RNA polymerase II (RNAPII) transcribes all of the cell protein-coding messenger RNAs (mRNAs) as well as some small nuclear RNAs (snRNAs). RNA polymerase III (RNAPIII) is dedicated to the transcription of a collection of genes whose main common feature is that they encode structural or catalytic RNAs (tRNAs, 5S RNA, snRNA) that are components of protein synthesis, splicing, and tRNA processing apparatuses. It is believed that this triplication event provided the eukaryotic cell with a greater flexibility toward energy-consuming cellular functions such as ribosome synthesis, as well as with more sophisticated means for the regulation of gene expression.Prokaryotic and eukaryotic RNA polymerases are multisubunit enzymes that are evolutionarily related to each other through their largest and second-largest subunits (Ebright 2000;Cramer 2002). The largest subunit (≈160 kDa: ␤Ј; A; RPA1; RPB1; RPC1) contains eight regions conserved in order and sequence (A to H), while the second-largest subunit (≈150 kDa: ␤; B; RPA2; RPB2; RPC2), contains nine such regions (A to I) (Allison et al. 1985;Sweetser et al. 1987). Although the role of these conserved domains is not yet fully understood, the structure determination of RNAPII suggests that they cooperate in the formation of a single fold cleft containing the active site of the enzyme (Cramer et al....

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A universally conserved region of the largest subunit participates in the active site of RNA polymerase III.

Cited by 71 publications

References 46 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

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

Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis

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