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.