DNA-dependent RNA

The mode of action of the naphthoquinoid ansamacroHdes was estabHshed through studies using the tifamycins and streptovaricins (84,141,257,258). The ansamacroHdes inhibit bacterial growth by inhibiting RNA synthesis. This is accompHshed by forming a tight complex with DNA-dependent RNA polymerase. This complex is between the ansamacroHde and the P-unit of RNA polymerase. The formation of the complex inhibits the initation step of RNA synthesis (259,260). The ansamacroHdes form no such complex with mammalian RNA polymerase and thus have low mammalian toxicity. 

DNA-Dependent RNA Polymerase Initiates Transcription at a Distinct Site, the Promoter... 

Mammalian Cells Possess Three Distinct Nuclear DNA-Dependent RNA Polymerases... 

The properties of mammahan polymerases are described in Table 37-2. Each of these DNA-dependent RNA polymerases is responsible for transcription of dif-... 

Table 37-2. Nomenclature and properties of mammalian nuclear DNA-dependent RNA polymerases.

One peptide toxin from the mushroom Amanita phalhides, a-amanitin, is a specific differential inhibitor of the eukaryotic nuclear DNA-dependent RNA polymerases and as such has proved to be a powerful research tool (Table 37-2). a-Amanitin blocks the translocation of RNA polymerase during transcription. 

Figure 37-7. Transcription elements and binding factors in the herpes simplex virus thymidine kinase ffW gene. DNA-dependent RNA polymerase II binds to the region of the TATA box (which is bound by transcription factor TEND) to form a multicomponent preinitiation complex capable of initiating transcription at a single nucleotide (+1).The frequency of this event is increased by the presence of upstream c/s-acting elements (the GC and CAAT boxes). These elements bind frans-acting transcription factors, in this example Spl and CTF (also called C/EBP, NF1, NFY). These cis elements can function independently of orientation (arrows).

There are three distinct nuclear DNA-dependent RNA polymerases in mammals RNA polymerases I, II, and III. These enzymes control the transcriptional function—the transcription of rRNA, mRNA, and small RNA (tRNA/5S rRNA, snRNA) genes, respectively. 

A second example of the differences between important biomolecules in archaebacteria and eubacteria is their DNA-dependent RNA polymerase. The enzyme found in archaea resembles that in eukaryotes more than it does those in bacteria ... 

To establish whether rifaximin, like the other members of the rifamycin family [36, 58], specifically inhibits bacterial RNA synthesis the effect of this antibiotic as well as that of rifampicin and chloramphenicol on RNA (via 3H-uridine incorporation), DNA (via 3H-thymidine incorporation) and protein (via 35S-methionine incorporation) synthesis was studied in growing cultures of Escherichia coli [59], While chloramphenicol reduced protein synthesis, both rifaximin and rifampicin inhibited RNA synthesis in a concentration-dependent fashion. In contrast, none of them affected 3H-thymidine incorporation into DNA. These data suggest that rifaximin, like rifampicin, inhibits RNA synthesis by binding the (3 subunit of the bacterial DNA-dependent RNA polymerase [60],... 

Development of resistance to rifaximin may be similar to that of rifampicin, which is primarily due to a chromosomal single-step alteration in the drug target, the DNA-dependent RNA polymerase [65, 66]. This differs from... 

Umezawa H, Mizuno S, Yamazaki H, Nitta K Inhibition of DNA-dependent RNA synthesis by rifamycins. J Antibiot (Tokyo) 1968,21 234-236. 

The mechanism of action of rifaximin depends on the inhibition of DNA-dependent RNA polymerase of the target microorganisms, leading to the suppression of initiation of chain formation in RNA synthesis. 

Abbreviations. a-M, a-mannosidase AP, acid phosphatase as-ni-ATPase, anion-stimulated, nitrate-inhibitable ATPase CCR, NAD(P)H-dependent cytochrome oreduc-tase cs-vi-ATPase, cation-stimulated, vanadate-inhibitable ATPase, CAT, catalase GS 1/11, glucan synthase 1 or 11 IDPase, inosine diphosphatase cs-PPase, cation-stimulated pyrophosphatase RNA polymerase, DNA-dependent RNA polymerase TP-25, 25 kDa tonoplast integral protein. 

Degradation of RNA DNA-dependent RNA polymerase Transcription factors RNA processing Other Translation... 

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

RNA is synthesized by a DNA-dependent RNA polymerase (uses DNA as a template for the synthesis of RNA). Important terminology used when discussing transcription is illustrated in Figure 1-3-2. 

The enzymes catalysing the synthesis of RNA, using DNA as a template, are known as DNA-dependent RNA polymerases. The term dependency indicates a requirement for a template. The RNA polymerases synthesise RNA in the 5 to 3 direction in formation of the phosphodiester linkage. This requires that the DNA template is read in the 5 to 3 direction (see below for explanation). 

Rifamycin SV (133, Fig. 23) is a naturally occurring macrocycle isolated from Nocardia mediterranei by Senti, Greco and Ballotta in 1959. It shows a high in vitro antibiotic activity through inhibition of DNA-dependent RNA polymerase, but bioavailability is low due to its poor water solubility. The semisynthetic derivative rifampicin (135) displays markedly higher water solubility and in vivo activity. We chose 3-formylrifamycin (134) as model macrocycle for the possibility of binding it to solid phase by hydrazone bond formation in a manner similar to rifampicin (Fig. 24). 

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

This consists of DNA-dependent RNA polymerase II and basal transcription factors (TFIIX, X = A - H). First, the basal factor TFIID binds to the promoter. TFIID, a large complex of numerous proteins, contains TATA boxbinding protein (TBP) and so-called TAFs (TBP-associated factors). The polymerase is attached to this core with the help of TFIIB. Before transcription starts, additional TFs have to bind, including TFIIH, which has heli-case activity and separates the two strands of DNA during elongation. In all, some 35 differ-... 

Rifampicin exhibits an antibacterial effect by inhibiting RNA synthesis. It inhibits DNA-dependent RNA polymerase by preventing the initial development of the chain, not by removing it. Rifampicin does not bind with the RNA polymerase nucleus of mam-maliam cells, and does not have an effect on corresponding RNA synthesis. It can inhibit mitochondrial RNA synthesis however, concentrations required for this exceed those necessary for synthesizing RNA by several hundred times. 

Pharmacology Rifampin inhibits DNA-dependent RNA polymerase activity in susceptible cells. Specifically, it interacts with bacterial RNA polymerase, but does not inhibit the mammalian enzyme. Cross-resistance has only been shown with other rifamycins. Rifampin at therapeutic levels has demonstrated bactericidal activity against intracellular and extracellular Mycobacterium tuberculosis organisms. Pharmacokinetics ... 

Pharmacology Rifabutin, an antimycobacterial agent, is a semisynthetic ansamycin antibiotic derived from rifamycin S. It is not known whether rifabutin inhibits DNA-dependent RNA polymerase in Mycobacterium avium or in Mycobacterium... 

Rifapentine (Priftin) [Antibiotic/Antitubercular Agent] Uses Pulm TB Action X DNA-dependent RNA polymerase. Dose Intensive phase ... 

---