Initiation of RNA molecules by purified Escherichia coli RNA polymerase

Summary The kinesties of appearance of, and the distribution among, the four bases of the chain-initial nucleotides (5-termini) of RNA chains synthesized in vitro under various conditions have been investigated. The results of this study have shown that when native T4 bacteriophage DNA is the template for the RNA polymerase, most chains start with a purine nucleotide. The ratio of ATP termini to GTP termini is independent of the reaction time and of the template/enzyme ratio in the reaction mixture. A similar preferential purine initiation was observed when denatured T4 is the template, but the ratio of ATP to GTP termini is reduced. All 5-termini of poly-AU chains synthesized on poly-dAT templates are ATP.The determination of the kinetics of initiation of RNA chains has allowed direct confirmation of some conclusions which had been inferred previously from sedimentation analyses of the RNA product. (1) Most RNA chains are initiated during a short period at the outset of the reaction. (2) Low concentrations of native DNA templates limit the number of RNA chains synthesized, not the rate of RNA chain growth. (3) The maximum number of RNA molecules which can be synthesized on denatured DNA templates is severalfold larger than the maximum number which can be synthesized on the same weight of native DNA.     

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted.     

Relaxed mutants of Escherichia coli RNA polymerase

When Escherichia coli cells are treated with either polymixin or gramicidin at concentrations that block protein and RNA synthesis, they accumulate a significant amount of guanosine tetraphosphate ppGpp. Such accumulation occurs in stringent (relA+) as well as in relaxed (relA) strains and no guanosine pentaphosphate pppGpp is then detected within the cells. These observations suggest that polypeptide antibiotics elicit ppGpp formation through a mechanism different from the stringent control system triggered by amino acid starvation of bacteria. Experiments based on tetracycline action indicate, moreover, that the accumulation of ppGpp under polymixin or gramicidin treatment is connected with a strong restriction of the degradation rate of this nucleotide.