**2.5 Inhibition of microbial RNA synthesis**

Rifamycins inhibit DNA-dependent transcription by binding the DNA-bound and effectively transcribing RNA polymerase with a high affinity to the β-subunit (coded by rpoB). In the channel formed by the RNA polymerase-DNA complex, from which the newly synthesized RNA strand emerges, the β- subunit is located. Rifamycins clearly require that RNA synthesis has not progressed beyond two ribonucleotides being added; This is due to the drug molecule 's capacity to sterically inhibit the initialization of nascent RNA strands. It should be noted that rifamycins are not believed to work by blocking the RNA synthesis elongation stage, although a recently discovered class of RNA polymerase inhibitors (based on the CBR703 compound) could inhibit elongation by modifying the enzyme allosterically [34].

### **2.6 Inhibition of microbial metabolic pathways**

By competitively blocking the biosynthesis of tetrahydrofolate, which acts as a carrier of one-carbon fragments and is required for the ultimate synthesis of DNA, RNA and bacterial cell wall proteins, trimethoprim and sulfonamides interfere with folic acid metabolism in the microbial cell. Bacteria and protozoan parasites typically lack a transport mechanism in order to extract preformed folic acid from their host, unlike mammals [29]. Most of these species, while some are capable of using exogenous thymidine, must synthesize folic acid, circumventing the need for metabolism of folic acid. The conversion of pteridine and p-aminobenzoic acid (PABA) to dihydrofolic acid by the pteridine synthetase enzyme is competitively inhibited by sulfonamides. Sulfonamides have a greater affinity for pteridine synthetase than for PABA. Trimethoprim has a huge affinity (10,000 to 100,000 times greater than that of the mammalian enzyme) for bacterial dihydrofolate reductase; it inhibits tetrahydrofolate synthesis when bound to this enzyme [29].
