Novel layers of RNA polymerase III control affecting tRNA gene transcription in eukaryotes

Abstract

RNA polymerase III (Pol III) transcribes a limited set of short genes in eukaryotes producing abundant small RNAs, mostly tRNA. The originally defined yeast Pol III transcriptome appears to be expanding owing to the application of new methods. Also, several factors required for assembly and nuclear import of Pol III complex have been identified recently. Models of Pol III based on cryo-electron microscopy reconstructions of distinct Pol III conformations reveal unique features distinguishing Pol III from other polymerases. Novel concepts concerning Pol III functioning involve recruitment of general Pol III-specific transcription factors and distinctive mechanisms of transcription initiation, elongation and termination. Despite the short length of Pol III transcription units, mapping of transcriptionally active Pol III with nucleotide resolution has revealed strikingly uneven polymerase distribution along all genes. This may be related, at least in part, to the transcription factors bound at the internal promoter regions. Pol III uses also a specific negative regulator, Maf1, which binds to polymerase under stress conditions; however, a subset of Pol III genes is not controlled by Maf1. Among other RNA polymerases, Pol III machinery represents unique features related to a short transcript length and high transcription efficiency.

Keywords: Pol III; TFIIIC; polymerase assembly; tRNA; transcription elongation; transcription termination read-through.

Figure 1.

Pol III biogenesis. Based on the relatively well-studied analogous process for prokaryotic RNA polymerase, it is postulated that the assembly of yeast Pol III starts with the formation of the AC19/AC40 subcomplex, probably together with the small ABC10β/ABC10α subunits, which then binds the second-largest catalytic subunit C128. The stable subcomplex C128/AC40/AC19/ABC10β/ABC10α binds the Rbs1 factor via AC40 and AC19. In a parallel step, the second major assembly intermediate is formed by the largest subunit, C160, and the ABC27 and ABC23 subunits incorporated with the help of Bud27. Pol III core is formed by joining of the two subcomplexes. Then the peripheral subunits are added as Pol III-specific subcomplexes (once the Pol III holoenzyme is assembled, Pol III subunits are presented in grey, for clarity). Gpn2, Gpn3 and Ssa4 presumably participate in later steps of Pol III biogenesis, and Iwr1 acts downstream of the GTPases and Ssa4. According to the presented model, Pol III complex is assembled in the cytoplasm prior to the nuclear import. It is also conceivable that only the core complex is formed in the cytoplasm and the peripheral subunits join it in the nucleus, as discussed in the text. Pol III is imported into the nucleus via the nuclear pore complex (NPC), probably together with the adaptors and assembly factors. The transport/assembly factors dissociate from Pol III and are exported back to the cytoplasm; Rbs1 and Ssa4 are exported, respectively, in Crm1- and Msn5-dependent manner.

Figure 2.

Historical view of Pol III-transcribed genes. The timeline presents approximate dates of identification of S. cerevisiae Pol III-transcribed loci as well as loci occupied only by TFIIIC. Numbers in superscript refer to the respective publications.

Figure 3.

Uneven distribution of Pol III on transcription units. (a) Pol III distribution pattern, identified by CRAC method, across most genes, with a high peak of nascent transcript density over the 5′-end of the transcription unit and a weaker peak before the 3′-end of mature tRNA (intron-less tRNA gene is shown). Read-through (RT) of termination signal is observed on many tRNA genes, typically extending 50–200 nt beyond the expected canonical termination site. (b) Localization of A- and B-boxes of the bipartite internal promoter, and termination site (T_n) in a tRNA-encoding gene (tDNA). (c) The τA and τB modules of TFIIIC factor binding the A- and B-boxes. Regions of postulated transient pausing of Pol III correspond to the TFIIIC binding sites.

Figure 4.

Regulation of Pol III transcription by Maf1. (a) Under favourable growth conditions, Maf1 is inactivated by phosphorylation. CK2 kinase phosphorylates Maf1 and also TFIIIB initiation factor associated in the promotor region stimulating Pol III transcription. (b) Upon shift to repressive conditions, CK2 dissociates from the Pol III complex. Dephosphorylated Maf1 binds directly to Pol III complex and weakens interaction of C34 with the Brf1 subunit of the TFIIIB initiation factor, and thereby impairs Pol III recruitment to promoters reducing transcription for nearly all tRNA genes. However, a subset of housekeeping tRNA genes marked in green exhibits low responsiveness to Maf1.