Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Trpby aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two D-aminoacylated tRNAs behaved as substrates of purified E. coli D-Tyr-tRNA Tyr deacylase. These results indicate that an unexpected high number of D-amino acids can impair the bacterium growth through the accumulation of D-aminoacyl-tRNA molecules and that D-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 produces D-Tyr-tRNA Tyr , and which, like E. coli, possesses a D-Tyr-tRNA Tyr deacylase activity. In this case, inhibition of growth by the various 19 D-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of D-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 two D-aminoacyl-tRNAs are substrates of a same D-aminoacyl-tRNA deacylase. In the case of D-Glu production, a racemase or a transaminase is involved, depending on the bacterium. Small amounts of D-amino acids can also possibly appear as side products of various biosynthetic pathways. Conversion of the L-to the D-stereoisomer of tryptophan was observed in the presence of tryptophan synthase (11,12). Similarly, in the case of methionyl-tRNA synthetase, a weak catalysis of ␣-carbon hydrogen-deuterium exchange of L-methionine was evidenced in vitro (13). Such an exchange suggests possible conversion of L-methionine into D-methionine at the surface of an amino acid-binding enzyme. As discussed earlier (14), D-tyrosine might arise at the step of the addition of an amino group to 4-hydroxyphenylpyruvate. Moreover, D-amino acids are likely to be nonspecifically formed as side reaction products in the presence of pyridoxal phosphate-containing enzymes or of pyridoxal phosphate alone (15, 16).If externally added, D-amino acids exert toxicity toward many organisms (1,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Possible causes of this toxicity are multiple. For instance, in the case of Escherichia coli, D-amino acids can be lethal because they are erroneously incorporated in peptidoglycan (23,25,26). In the case of Bacillus subtilis, strains capable of efficiently pumping D-tyrosine have been described. The growth of such strains is decreased upon addition of this D-amino acid to the culture medium (19). Inhibition of prephenate dehydrogenase and the consequent curtailment of L-tyrosine biosynthesis may account for this behavior (18). However, incorporation of D-tyrosine into proteins could be evidenced with the above B. subtilis strains (18).In agreement with this observation, several studies indicateTyr formation in E. coli (27-29) as well as in Saccharomyces cerevisiae (14). Moreover, to recycle tRNA Tyr esterified by D-tyrosine and/or to minimize ...

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.

Gene transcription is highly regulated. Altered transcription can lead to cancer or developmental diseases. Mediator, a multisubunit complex conserved among eukaryotes, is generally required for RNA polymerase II (Pol II) transcription. An interaction between the two complexes is known, but its molecular nature and physiological role are unclear. We identify a direct physical interaction between the Rpb3 Pol II subunit of Saccharomyces cerevisiae and the essential Mediator subunit, Med17. Furthermore, we demonstrate a functional element in the Mediator-Pol II interface that is important for genome-wide Pol II recruitment in vivo. Our findings suggest that a direct interaction between Mediator and Pol II is generally required for transcription of class II genes in eukaryotes.

Thus, the S2P polymerase plays a specific, regulatory role in cell differentiation through the induction of ste11.

RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling.Transcription occurs in the crowded context of the nucleus in which genes are wrapped in chromatin. The first step in gene expression involves the modification and/or the remodeling of repressive chromatin by specialized complexes. For polymerase II (Pol II)-transcribed genes, these steps are followed by the recruitment of Mediator, the general transcription factors (GTFs) and the Pol II itself, although in an order that can vary from one promoter to another (9, 34). The pathway leading from silent chromatin to transcription by Pol I and Pol III has not been studied as thoroughly but is globally similar, with an additional contribution of cognate GTFs. In yeast and human cells, the Pol III-specific transcription factor TFIIIC has been found to be required for the proper nucleosomal organization of Pol III genes (4, 23, 32). In the case of Pol I transcription, the mammalian termination factor TTF-I is able to activate transcription by promoting chromatin remodeling in synergy with ATP-dependent cofactors in vitro (24). Transcription initiation is not the only step at which chromatin might interfere with transcription. Nucleosomes residing in the transcribed region can inhibit the movement of RNA polymerases during elongation. To contend with this, the FACT complex helps human Pol II transcribe through nucleosome-induced blocks (28, 38). These observations suggest that factors that relieve the repressive effect of nucleosomes might act in conjunction with the transcription machinery at the successive stages of the transcription cycle.The repressive effect of nucleosomes is overcome by two cooperative mechanisms. The first involves the covalent modification of the histones, including the acetylation of specific histone tail lysines by acetyl transferases (...

RNA polymerase (Pol) III synthesizes the tRNAs, the 5S ribosomal RNA and a small number of untranslated RNAs. In vitro, it also transcribes short interspersed nuclear elements (SINEs). We investigated the distribution of Pol III and its associated transcription factors on the genome of mouse embryonic stem cells using a highly specific tandem ChIP-Seq method. Only a subset of the annotated class III genes was bound and thus transcribed. A few hundred SINEs were associated with the Pol III transcription machinery. We observed that Pol III and its transcription factors were present at 30 unannotated sites on the mouse genome, only one of which was conserved in human. An RNA was associated with >80% of these regions. More than 2200 regions bound by TFIIIC transcription factor were devoid of Pol III. These sites were associated with cohesins and often located close to CTCF-binding sites, suggesting that TFIIIC might cooperate with these factors to organize the chromatin. We also investigated the genome-wide distribution of the ubiquitous TFIIS variant, TCEA1. We found that, as in Saccharomyces cerevisiae, TFIIS is associated with class III genes and also with SINEs suggesting that TFIIS is a Pol III transcription factor in mammals.

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 vitro conditions, proceeds at a rate of 6 s-1 with a Km value for the substrate equal to 1 microM. Cell growth is sensitive to interruption of the yihZ 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 the D-Tyr-tRNATyr deacylase function in the detoxification of cells, similar genes can be recognized in Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, mouse, and man.