Substrate properties of C'-methylnucleoside triphosphates in a reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase

2 theta-C-methyl substituted and phosphonate analogs of UTP were prepared and together with the synthesized earlier 3'-C-methyl-UTP were investigated in the RNA synthesis reaction catalysed by Escherichia coli RNA-polymerase. Substrate properties of UTP analogs were studied in the presence of all natural triphosphates, in the absence of UTP and under conditions of soil substrate reaction. It was shown that UTP(3'CH3) is incorporated into the RNA chain and terminates further RNA elongation. Another analog UTP (2'CH3) may substitute natural UTP in RNA synthesis, but the effectivity of its incorporation is diminished. Phosphonate analog UTP(5'CH2) is a pseudoterminator of RNA synthesis. The conformational analysis of 2'- and 3'C-methylnucleosides by force-field method of calculation was carried out in order to find energetically forbidden conformations of these analogs due to the collision of bulky methyl group and a heterocyclic base. An attempt was made to fix the conformation of the substrate during its enzymatic transformation.     

5'-C-methylnucleoside triphosphates in the reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase

It was shown that RNA-polymerase is able to discriminate diastereoisomers of 5'-methyl-substituted analogs of ribonucleoside triphosphates (rNTP). Under conditions of soil substrate reactions when the analog is added to the presynthesized ternary complexes, D-allo- and L-talo-stereoisomers incorporate into RNA 100 and 1000 times, respectively, less effectively, then the natural rNTP. The effectivities of incorporation of other 2'- and 3'-substituted analogs of rNTP were measured under the same conditions and compared with that for 5'-Me-rNTP. It was shown also that RNA-polymerase does not support long-chain RNA synthesis from 5'-Me-rNTP in the absence of natural rNTP. No more then two analog residues can be attached to the 3'-end of the presynthesized RNA under such conditions. Addition of one natural rNTP to this reaction mixture results in the synthesis of long alternating RNA containing D-allo-stereoisomer and natural rNTP residues. In the case of L-talo-stereoisomer RNA elongation is not inhibited, if the distance between the analog residues in the RNA chain is not shorter then five nucleotide residues. The rate of pyrophosphorolysis from the RNA of the analogs studied was the same as for the natural rNTP residues.