Structure–function analysis of RNA polymerases I and III

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Recent advances in elucidating the structure of yeast Pol I and III are based on a combination of X-ray crystal analysis, electron microscopy and homology modelling. They allow a better comparison of the three eukaryotic nuclear RNA polymerases, underscoring the most obvious difference existing between the three enzymes, which lies in the existence of additional Pol-I-specific and Pol-III-specific subunits. Their location on the cognate RNA polymerases is now fairly well known, suggesting precise hypotheses as to their function in transcription during initiation, elongation, termination and/or reinitiation. Unexpectedly, even though Pol I and III, but not Pol II, have an intrinsic RNA cleavage activity, it was found that TFIIS Pol II cleavage stimulation factor also played a general role in Pol III transcription.

Introduction

Three nuclear RNA polymerases (Pol) operate in all eukaryotes examined so far. Whilst Pol II produces all mRNAs and many non-coding RNAs, and thus transcribes most of the nuclear genome, it contributes to less than 10% of the total RNA present in growing cells. Pol I is specialised in the synthesis of the abundant pre-rRNA precursor of the three largest rRNAs, and Pol III makes all tRNAs and the 5S rRNA, along with various short, non-translated RNAs [1]. Pol I and III typically account for some 75% and 15% of the whole transcription output, respectively, in fast growing yeast cells, and thus play a key role in the control of cell growth [2]. Structural studies on eukaryotic Pols were so far focused on yeast Pol II (reviewed by Cramer et al., in this issue and reference [3]), but recent progress made in elucidating the structure of yeast Pol I and Pol III now pave the way to an in-depth comparison of the three nuclear transcription systems.

Section snippets

Structural model of Pol III and function of the subunits

Yeast Pol I, II and III have related subunit compositions, with twelve homologous or identical core subunits shared by all three Pols (Table 1). All these subunits have archaeal paralogues [4, 5, 6•] and five of them, including the two largest ones, are also homologous to the bacterial β′βα2ω core Pol [7]. Yeast Pol III also contains five enzyme-specific subunits (Rpc31, Rpc34, Rpc37, Rpc53 and Rpc82), present in all eukaryotic lineages. This complex structure presumably reflects the

Structural model of Pol I: the role of Pol-I-specific subunits

Half of the fourteen yeast Pol I subunits are shared with Pol III, and this common organisation also exists in the other eukaryotic lineages, with minor exceptions due to the Rpb5, Rpb6 and Rpb10 isoforms existing in plants and/or trypanosomes [15, 24]. A 12 Å electron microscopy (EM) envelope of the complete 14-subunit yeast Pol I has been combined with a detailed crystal structure of the Rpa14/Rpa43 stalk [25••]. As expected, differences to Pol II or III are mostly due to the Pol-I-specific

Pol I recruitment and transcription initiation

As in the Pol II and Pol III cases, a pre-initiation complex that contains the TBP protein is needed to recruit Pol I to its promoter DNA. A major difference, however, is that Pol I is only targeted to its pre-initiation complex when bound to a specialised protein, Rrn3, which together form an Rrn3–Pol I structure competent for transcription initiation [29]. The formation of the Rrn3/Pol I complex involves a direct interaction between Rrn3 and the Rpa43 component of the Pol I stalk. An

The atypical Pol I of trypanosomes

Pol I subunits are generally less conserved than those of Pol II or III. Thus, sequence homology to Rpa14 is restricted to S. pombe and other Ascomycetes, and Rpa34-like sequences are limited to fungi, animals and amoebae, which belong to the monophyletic lineage of Eukaryotes known as Unikonts [27•, 28]. The evolutionary divergence of Pol I is especially strong in Trypanosomes where Pol I, beyond its canonical role in rDNA transcription, also synthesises the highly abundant mRNAs of procyclin

New roles for an old protein: TFIIS functions in Pol II and Pol III transcription

TFIIS is a highly conserved Pol-II-elongation factor that stimulates the RNA cleavage activity of Pol II, therefore acting as an elongation factor in vitro [39]. Moreover, there is mounting evidence that it also acts at some early stage of transcription initiation [40, 41, 42, 43, 44]. Its crystal structure, in association with Pol II (Figure 2), shows that TFIIS reaches the active site through the bottom pore of Pol II, which brings a highly conserved C-terminal zinc loop very close to the

Concluding remarks

The structural models now available for yeast Pol I and Pol III have allowed a much better comparison of the three eukaryotic RNA polymerases and, in particular, have refined our understanding of the transcriptional roles by the enzyme-specific subunits and by the conserved stalk fold. Future work will hopefully bring these structures to the atomic level of resolution now only available for Pol II. By contrast, very little is known of the structure and spatial organisation of the pre-initiation

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We dedicate this review to André Sentenac, as a tribute to his important contribution to Pol I and Pol III studies. We also thank past and present co-workers, too numerous to be individually quoted here, and apologize to colleagues whose work was not specifically discussed owing to stringent space limitations. Work in our laboratory was supported by the National Agency for Research, the Association pour la Recherche sur le Cancer, the Association Française contre les Myopathies and the Région

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