Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae.

Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

Relative affinities of nucleotide substrates for the yeast tRNA gene transcription complex.

Apparent Michaelis constants for nucleotides in transcription of yeast tRNA gene by homologous RNA polymerase III with auxiliary protein factors, were found to be remarkably higher in initiation than in elongation of RNA chain. This supports presumptions regarding topological similarities between catalytic centers of bacterial and eukaryotic RNA polymerases.

Role of metal ions in the assembly and decay of the transcription initiation complex on tRNA gene in yeast extracts.

Studies on formation of the transcription initiation complex on the tRNA(Tyr) gene in yeast extracts with the use of Sarkosyl-challenge assay proved that a single magnesium ion is indispensable for the complex assembly in the absence of nucleotides. Other cations do not substitute for Mg2+. The optimal KC1 concentration for transcription of the gene by the assembled initiation complex was found to be twice as high as that for the assembly process and close to that observed previously for the TFIIIC (or tau) factor dissociation from the promoter. The preformed complex remained stable for several hours at room temperature and its decay was not influenced by ionic strength. The data seem to support the notion that the TFIIIC factor is used only for assembly of the initiation complex and is not necessarily involved in the subsequent steps of transcription.

Substrate selection by RNA polymerase from E. coli. The role of ribose and 5'-triphosphate fragments, and nucleotides interaction.

Steady-state kinetic studies of the rifampicin-effected abortive initiation of transcription by E. coli RNA polymerase (EC 2.7.7.6) on the A1 T7 phage promoter were carried out with the use of ATP, UTP and a number of their appropriately modified analogues. The kinetic parameters KiA, KmB, Ki and KsB characterizing the affinity of the substrates and inhibitors of the reaction to the initiation and elongation sites of the enzyme:promoter and the enzyme:promoter:nucleoside triphosphate complexes were determined therefrom. Their comparative analysis indicated that 1) the triphosphate chain of the initiating purine nucleoside triphosphate interacts with some protein acceptor groups through the alpha- and beta-phosphate residues; the phosphates are engaged in binding of nucleoside triphosphates at the elongation site in the absence of the primer nucleotide; 2) the ribose 2'-OH of the elongating nucleotide, but neither of the ribose hydroxyl groups of the initiating nucleotide, participate in substrate recognition by protein receptors; 3) either substrate, ATP or UTP, bound to the initiation complex increases by about the same factor (greater than or equal to 10) the affinity of the other to its binding site; 4) the 3'-OH of the primer nucleotide and the gamma-phosphate of the elongating nucleotide are involved in the synergistic interaction of the substrates; alpha- and beta-phosphates of the elongating nucleotide, bound to some protein receptors, also contribute to this process. It is postulated that the interaction of substrates is mediated through an Mg2+ ion, known to be required for binding of the substrates in the elongation site, and a minimal molecular model of a PuoTP:Mg (II): nucleoside triphosphate chelate complex in the catalytic centre of the transcription initiation open complex is proposed.

Properties of P3 esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli.

P3-[(2,4-Dinitrophenyl)amino]ethyl (DNPNHEt) and P3-methyl phosphate esters of nucleoside 5'-triphosphates have been synthesized. Their properties as substrates in the initiation and elongation steps of transcription have been examined by using RNA polymerase from Escherichia coli and poly[d(A-T)] or T7 DNA as templates. It is shown that transcription can be initiated by ATP-EtNHDNP and that 2,4-dinitrophenyl residues are incorporated at the 5' end of the RNA molecules. Steady-state kinetic experiments of abortive initation on promoters A1 and A3 of T7 DNA revealed that ATP-EtNHDNP, ADP-EtNHDNP, and ATP-OCH3 have lower Km values and markedly reduced Vmax values compared to those of ATP. The two classes of esters, NTR-EtNHDNP and NTP-OCH3, were found to differ regarding their utilization as substrates for elongation. Both ATP-OCH3 and UTP-OCH3 are substrates for transcription. However, only the pyrimidine derivatives of NTP-EtNHDNP are elongation substrates which release DNPNHEt-PP upon utilization. This dramatic difference between the purine and pyrimidine derivatives of NTP-EtNHDNP reflects a selective process in the transcriptional complex for purines and pyrimidines.

The properties of ATP-analogs in initiation of RNA synthesis catalyzed by RNA polymerase from E coli.

Various base and sugar modified derivatives of ATP and UTP were used as substrate analogs for the steady state initiation reaction ATP+UTP=pppApU and the single step addition reaction ApC+ATP=ApCpA. These reactions were carried out by E. coli RNA polymerase on T7 DNA in the presence of rifampicin. The steady state kinetic parameters of the analogs, either as substrates or inhibitors, were determined. On the basis of the obtained results it is concluded that purine NTP s in initiation require anti-conformation about the glycosidic bonds as well as gauche-gauche conformation of the C(4')-C(5') bonds. The latter conformation is also a prerequisite for substrates in elongation, whereas strict anti-conformation of glycosidic bonds is not.

Primed abortive initiation of RNA synthesis by E. coli RNA polymerase on T7 DNA. Steady state kinetic studies.

Ternary complexes of T7 DNA, RNA polymerase and the antibiotic rifampicin carry out the promoter specific abortive initiation when dinucleoside monophosphates were employed as primers. Primed abortive initiation, leading to synthesis of trinucleoside diphosphates, only occured with combinations of primers and substrates complementary to a promoter region of 8 base pairs centered around the origin of transcription. The steady state kinetics of three abortive initiations at T7 promoter A3 were studied in detail. The reactions appeared to be truly ordered. Affinity constants, maximal velocities and elementary step rate constants were thus obtained. The stimulation by dinucleoside monophosphate primers is brought about by positively effecting the function of the substrate site rather then by their higher affinity to the primer site of the transcriptional complex.