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

Abstract

SUMMARYTernary 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.

NanoRNAs Prime Transcription Initiation In Vivo

“…Thus, the data presented in Figure 2 indicate that 2- to 4-nt RNAs that are complementary to the DNA template can effectively compete with NTPs for use as primers by P. aeruginosa RNAP during transcription initiation in vitro provided the 5′ end of the RNA is complementary to sequences between positions −3 and +1 and the 3′ end is complementary to position +1, +2 or +3. As anticipated, these findings are consistent with prior in vitro analysis of E. coli RNAP (Di Nocera et al, 1975; Grachev et al, 1984; Hoffman and Niyogi, 1973; Minkley and Pribnow, 1973; Ruetsch and Dennis, 1987; Smagowicz and Scheit, 1978). …”

“…Prior studies with E. coli RNAP indicate that use of 2- to 4-nt RNAs to prime transcription initiation in vitro can, in certain cases, result in transcription start site (TSS) shifting to positions −3, −2, or −1 (Di Nocera et al, 1975; Grachev et al, 1984; Hoffman and Niyogi, 1973; Minkley and Pribnow, 1973; Ruetsch and Dennis, 1987; Smagowicz and Scheit, 1978). In particular, studies with E. coli RNAP suggest that 2- to 4-nt RNAs are effective primers during transcription initiation in vitro provided the 5′ end of the RNA is complementary to sequences between positions −3 and +1 and the 3′ end is complementary to position +1, +2 or +3.…”

“…However, it is well established that prokaryotic and eukaryotic RNA polymerase (RNAP) can utilize 2- to ~8-nt RNAs to prime transcription initiation in vitro (Chamberlin and Ring, 1973; Di Nocera et al, 1975; Grachev et al, 1984; Hoffman and Niyogi, 1973; Learned and Tjian, 1982; Minkley and Pribnow, 1973; Niyogi and Stevens, 1965; Ruetsch and Dennis, 1987; Samuels et al, 1984; Smagowicz and Scheit, 1978). Nevertheless, whether 2- to ~8-nt RNAs can also prime transcription initiation in vivo has not previously been tested.…”

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

“…Through this procedure, different RNAP-promoter initially transcribed complex states (RP ITC≤i , i – the maximal length of transcribed RNA, Fig. S4E ) 95 96 are visited at which the DNA bubble is scrunched by RNAP 97 98 99 allowing downstream DNA bases to melt and serve as reading template for polymerization of RNA.…”

Generality of the Branched Pathway in Transcription Initiation byEscherichia coli RNA Polymerase

“…Using this promoter, the order of binding of substrates (2) and the rate of incorporation of single nucleotides were determined (2,15) as well as the characteristics of abortive synthesis (17,18). The concepts of promoter clearance (16) and of initial transcribing complex/initial elongating complex (19) were established by experiments on promoters including T7A1, and the translocational movement of RNA polymerase away from this promoter was systematically studied (20,21).…”