RNA Initiation with Dinucleoside Monophosphates during Transcription of Bacteriophage T4 DNA with RNA Polymerase of Escherichia coli

Abstract

The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: (i) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. (ii) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5'-guanosyl dinucleoside monophosphates. (iii) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. (iv) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. (v) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 "early" type. (vi) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of 3H-labeled stimulatory dinucleoside monophosphates support the above conclusions.The discovery of the sigma (a) subunit of bacterial RNA polymerase added strength to the concept of positive control of transcription in vitro (1-3). a-Factor is a necessary requirement for accurate initiation of RNA synthesis by core RNA polymerase in vitro on certain phage DNA templates (4-7). Our studies with well-defined templates have shown that the stimulatory effect of o-factor depends on both the base composition and secondary structure of the template, as well as the concentration of ribonucleoside triphosphate, and that the same type of stimulatory effect is obtained with oligoribonucleotides complementary to the templates (8). Earlier studies by Niyogi and Stevens (9) with holo RNA polymerase and synthetic polyribonucleotide templates indicated that only complementary oligoribonucleotides with a free 3'-hydroxyl group are stimulatory and preferentially incorporated into the 5'phosphomonoester chain end of the product, hence acting as chain initiators. Gros et al. (10)

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.…”

NanoRNAs: A Class of Small RNAs That Can Prime Transcription Initiation in Bacteria

“…In vitro studies indicate that 2- to 4-nt RNAs that are complementary to the DNA template can efficiently compete with NTPs for use as primers during transcription initiation provided the 5′ end of the RNA base pairs with promoter sequences between positions −3 and +1 and the 3′ end of the RNA base pairs with promoter sequences between positions +1 and +3 11, 12, 19–23 . (Note that by convention the position where de novo transcription begins is designated +1.)…”

“…In particular, it was proposed that small RNA fragments derived from the processing of longer transcripts could serve as sequence-specific primers that stimulate transcription initiation from specific target promoters 8, 9 . The proposal that small RNA fragments could “activate” transcription in this manner was based upon the empirical observation that yields of RNA transcripts produced in vitro could, in certain cases, be increased by adding a small RNA primer to the transcription reactions 2, 3, 10, 11 . Nevertheless, in the ~35 years since this proposal was made, no evidence that small RNAs can actually prime transcription initiation in vivo has been presented.…”