Julie Soutourina

Mediator-Dependent Recruitment of TFIIH Modules in Preinitiation Complex

In vitro, without Mediator, the association of general transcription factors (GTF) and RNA polymerase II (Pol II) in preinitiation complexes (PIC) occurs in an orderly fashion. In this work, we explore the in vivo function of Mediator in GTF recruitment to PIC. A direct interaction between Med11 Mediator head subunit and Rad3 TFIIH subunit was identified. We explored the significance of this interaction and those of Med11 with head module subunits Med17 and Med22 and found that impairing these interactions could differentially affect the recruitment of TFIIH, TFIIE, and Pol II in the PIC. A med11 mutation that altered promoter occupancy by the TFIIK kinase module of TFIIH genome-wide also reduced Pol II CTD serine 5 phosphorylation. We conclude that the Mediator head module plays a critical role in TFIIH and TFIIE recruitment to the PIC. We identify steps in PIC formation that suggest a branched assembly pathway.

Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells.

In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNATyr with tyrosine. Resulting d-Tyr-tRNATyr can be hydrolyzed by ad-Tyr-tRNATyr deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encodingd-Tyr-tRNATyr deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate,d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNAAspand d-Trp-tRNATrp by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was establishedin vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNATyr deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation ofd-aminoacyl-tRNA molecules and thatd-Tyr-tRNATyr deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also producesd-Tyr-tRNATyr, and which, like E. coli, possesses a d-Tyr-tRNATyr deacylase activity. In this case, inhibition of growth by the various 19d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects ofd-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting twod-aminoacyl-tRNAs are substrates of a samed-aminoacyl-tRNA deacylase.

TFIIS elongation factor and Mediator act in conjunction during transcription initiation in vivo

The transcription initiation and elongation steps of protein-coding genes usually rely on unrelated protein complexes. However, the TFIIS elongation factor is implicated in both processes. We found that, in the absence of the Med31 Mediator subunit, yeast cells required the TFIIS polymerase II (Pol II)-binding domain but not its RNA cleavage stimulatory activity that is associated with its elongation function. We also found that the TFIIS Pol II-interacting domain was needed for the full recruitment of Pol II to several promoters in the absence of Med31. This work demonstrated that, in addition to its thoroughly characterized role in transcription elongation, TFIIS is implicated through its Pol II-binding domain in the formation or stabilization of the transcription initiation complex in vivo.

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

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.

Recruitment of P-TEFb (Cdk9-Pch1) to chromatin by the cap-methyl transferase Pcm1 in fission yeast.

Capping of nascent pre‐mRNAs is thought to be a prerequisite for productive elongation and associated serine 2 phosphorylation of the C‐terminal domain (CTD) of RNA polymerase II (PolII). The mechanism mediating this link is unknown, but is likely to include the capping machinery and P‐TEPb. We report that the fission yeast P‐TEFb (Cdk9‐Pch1) forms a complex with the cap‐methyltransferase Pcm1 and these proteins colocalise on chromatin. Ablation of Cdk9 function through chemical genetics causes growth arrest and abolishes serine 2 phosphorylation on the PolII CTD. Strikingly, depletion of Pcm1 also leads to a dramatic decrease of phospho‐serine 2. Chromatin immunoprecipitations show a severe decrease of chromatin‐bound Cdk9‐Pch1 when Pcm1 is depleted. On the contrary, Cdk9 is not required for association of Pcm1 with chromatin. Furthermore, compromising Cdk9 activity leads to a promoter‐proximal PolII stalling and sensitivity to 6‐azauracil, reflecting elongation defects. The in vivo data presented here strongly support the existence of a molecular mechanism where the cap‐methyltransferase recruits P‐TEFb to chromatin, thereby ensuring that only properly capped transcripts are elongated.

Functional characterization of the D-Tyr-tRNATyr deacylase from Escherichia coli.

The yihZ gene of Escherichia coli is shown to produce a deacylase activity capable of recycling misaminoacylated d-Tyr-tRNATyr. The reaction is specific and, under optimal in vitroconditions, proceeds at a rate of 6 s−1 with aK m value for the substrate equal to 1 μm. Cell growth is sensitive to interruption of theyihZ gene if d-tyrosine is added to minimal culture medium. Toxicity of exogenous d-tyrosine is exacerbated if, in addition to the disruption of yihZ, the gene of d-amino acid dehydrogenase (dadA) is also inactivated. Orthologs of the yihZ gene occur in many, but not all, bacteria. In support of the idea of a general role of thed-Tyr-tRNATyr deacylase function in the detoxification of cells, similar genes can be recognized inSaccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, mouse, and man.

Structure–function analysis of RNA polymerases I and III

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.

Formation of d-Tyrosyl-tRNATyr Accounts for the Toxicity of d-Tyrosine toward Escherichia coli

d-Tyr-tRNATyr deacylase cleaves the ester bond between a tRNA molecule and a d-amino acid. In Escherichia coli, inactivation of the gene (dtd) encoding this deacylase increases the toxicity of several d-amino acids including d-tyrosine, d-tryptophan, and d-aspartic acid. Here, we demonstrate that, in a Δdtd cell grown in the presence of 2.4 mm d-tyrosine, ∼40% of the total tRNATyr pool is converted into d-Tyr-tRNATyr. No d-Tyr-tRNATyr is observed in dtd+ cells. In addition, we observe that overproduction of tRNATyr, tRNATrp, or tRNAAsp protects a Δdtd mutant strain against the toxic effect of d-tyrosine, d-tryptophan, or d-aspartic acid, respectively. In the case of d-tyrosine, we show that the protection is accounted for by an increase in the concentration of l-Tyr-tRNATyr proportional to that of overproduced tRNATyr. Altogether, these results indicate that, by accumulating in vivo, high amounts of d-Tyr-tRNATyr cause a starvation for l-Tyr-tRNATyr. The deacylase prevents the starvation by hydrolyzing d-Tyr-tRNATyr. Overproduction of tRNATyr also relieves the starvation by increasing the amount of cellular l-Tyr-tRNATyr available for translation.

Nucleosome Depletion Activates Poised RNA Polymerase III at Unconventional Transcription Sites in Saccharomyces cerevisiae

RNA polymerase (pol) III, assisted by the transcription factors TFIIIC and TFIIIB, transcribes small untranslated RNAs, such as tRNAs. In addition to known pol III-transcribed genes, the Saccharomyces cerevisiae genome contains loci (ZOD1, ETC1-8) associated to incomplete pol III transcription complexes (Moqtaderi, Z., and Struhl, K. (2004) Mol. Cell. Biol. 24, 4118-4127). We show that a short segment of the ZOD1 locus, containing box A and box B promoter elements and a termination signal between them, directs the pol III-dependent production of a small RNA both in vitro and in vivo. In yeast cells, the levels of both ZOD1- and ETC5-specific transcripts were dramatically enhanced upon nucleosome depletion. Remarkably, transcription factor and pol III occupancy at the corresponding loci did not change significantly upon derepression, thus suggesting that chromatin opening activates poised pol III to transcription. Comparative genomic analysis revealed that the ZOD1 promoter is the only surviving portion of a tDNAIle ancestor, whose transcription capacity has been preserved throughout evolution independently from the encoded RNA product. Similarly, another TFIIIC/TFIIIB-associated locus, close to the YGR033c open reading frame, was found to be the strictly conserved remnant of an ancient tDNAArg. The maintenance, by eukaryotic genomes, of chromatin-repressed, non-coding transcription units has implications for both genome expression and organization.

Mutations of RNA polymerase II activate key genes of the nucleoside triphosphate biosynthetic pathways

The yeast URA2 gene, encoding the rate‐limiting enzyme of UTP biosynthesis, is transcriptionally activated by UTP shortage. In contrast to other genes of the UTP pathway, this activation is not governed by the Ppr1 activator. Moreover, it is not due to an increased recruitment of RNA polymerase II at the URA2 promoter, but to its much more effective progression beyond the URA2 mRNA start site(s). Regulatory mutants constitutively expressing URA2 resulted from cis‐acting deletions upstream of the transcription initiator region, or from amino‐acid replacements altering the RNA polymerase II Switch 1 loop domain, such as rpb1‐L1397S. These two mutation classes allowed RNA polymerase to progress downstream of the URA2 mRNA start site(s). rpb1‐L1397S had similar effects on IMD2 (IMP dehydrogenase) and URA8 (CTP synthase), and thus specifically activated the rate‐limiting steps of UTP, GTP and CTP biosynthesis. These data suggest that the Switch 1 loop of RNA polymerase II, located at the downstream end of the transcription bubble, may operate as a specific sensor of the nucleoside triphosphates available for transcription.